Molecular and Cellular Endocrinology, 86 (1992) 119-123 0 1992 Elsevier Scientific Publishers Ireland. Ltd. 0303.7207/92/$05.00
MOLCEL
119
02786
Calbindin-D,,
J. Krisinger,
mRNA is tightly regulated in the rat uterus J.L. Dann, W.D. Currie,
during the estrous cycle
E.B. Jeung and P.C.K. Leung
Deparrmrnt of Ohstrtrics and Gynaecology, Unirmity of’ British C‘olmha.
Key words: Calbindin;
Uterus;
Estrogen;
(Received
27 February
Estrous
cycle: Actin
1992; accepted
3 April
Vancoulscv, B.C. VSZ 4H4. Canada 1992)
Summary Calbindin-D,, (CaBP-9k) is a cytosolic calcium binding protein with a molecular weight of 9000. CaBP-9k is mainly expressed in intestine, uterus and placenta, with intestinal levels controlled by vitamin D and uterine levels controlled by estrogens. CaBP-9k mRNA levels were measured in rat uterus throughout the estrous cycle. On the morning of proestrus, estrus and diestrus animals were sacrificed. Serum 17/3-estradiol concentrations were determined using a radioimmunoassay. Whole uterus was used for preparation of total RNA. Northern blot analysis was performed to quantify CaBP-9k and p-actin mRNA. CaBP-9k levels were highest at proestrus, dropped lo-fold at estrus and were not detectable at diestrus. @Actin levels did not change significantly throughout the estrous cycle. Peak 17&estradiol concentrations coincided with maximum CaBP-9k mRNA expression at proestrus. Despite minimal concentrations of 17/3-estradiol at estrus, CaBP-9k mRNA was still present at 10% of the proestrus level. At diestrus, CaBP-9k mRNA was not detectable despite increasing 17P-estradiol. It is concluded that CaBP-9k is subject to 17j3-estradiol regulation during the estrous cycle. Correlation between CaBP-9k mRNA and 17P-estradiol levels indicates a lag period for CaBP-9k induction in diestrus following a rise in steroid hormone levels.
Introduction Calbindin-D,, (CaBP-9k) is a 9000 M, cytosolit calcium binding protein expressed predominantly in duodenum, placenta and uterus (Christakos et al., 1989). In the duodenum, it is transcriptionally controlled by 1,25dihydroxy-
Correspondence to: Dr. J. Krisinger. The Research Centre, Dept. of OB/GYN, No. 178 - 950 W. 28th Ave., Vancouver, B.C. V5Z 4H4, Canada. Tel. (604) 8752433; Fax 875.2496. This work was supported by grants from the British Columbia Health Research Foundation No. 7 (90-2). The Molly Towell Perinatal Research Foundation. and the Medical Research Council of Canada (No. MT771 1).
vitamin D,, the hormonal form of vitamin D (Kessler and DeLuca, 198.5; Brehier and Thomasset, 1990; Krisinger et al., 1990) and is involved in intestinal calcium absorption (Roche et al., 1986; Wasserman et al., 1989). The function of uterine CaBP-Ok remains unknown. It has been postulated that the protein is involved in controlling the intra-myometrial calcium concentration via estrogen-mediated hormonal control (Bruns et al., 1988). By virtue of its calcium binding activity, CaBP-9k may decrease intracellular free calcium concentration or act as a calcium controlled mediator of uterine activity similar to calmodulin (Kyozuka et al., 1987; Savineau et al., 1988). The uterine CaBP-9k is tissue specifically controlled
120
by 17@-estradiol (Ez) at the transcriptional level mediated by an estrogen response element near the CaBP-9k gent promoter (Darwish et al., 1990). Histochcmical studies have shown that CaBP-9k is cxprcsscd in myometrium and endometrium (Dclorme et al., lYX3; Warembourg et al., 1987; Bruns et al., 198X; Mathieu et al., lYX9). After ovariectomy, uterine mRNA encoding CaBP-9k falls below detection levels and expression can be restored by E2 administration (Darwish et al., lY90; L’Horset et al., 1990). The present study examined the possible influence of the estrous cycle on the expression pattern of CaBP-Yk in the ‘uterus. Material and methods Animals
Two- to three-month female Sprague-Dawley rats were fed rat chow and maintained under a 12 h light/dark cycle. On the morning of the experiment (1O:OO a.m.), a vaginal smear was performed and rats were sacrificed by decapitation after halothane/N,O anesthesia. Blood was collected and the abdominal cavity opened by a midventral incision. Phase of the estrous cycle was immediatcly determined by vaginal exfoliate cytology and confirmed by dissecting microscopic examination of ovarian ultrastructures and the macroscopic appearance of the uterus. Quantifi;catiotz of CaBP-9k
mRNA
Whole uteri were homogenized and total RNA prepared by the guanidinium/CsCl procedure (Glisin et al.. 1974; Chomczynski and Sacchi, lYX7). Standard Northern analysis was performed with 20 pg of total RNA. A 295 nucleotide rat cDNA probe (Darwish ct al., 1987) was synthesized by random primed synthesis using [cu” P]dCTP. After stringent hybridization and washing, the blots were subjected to autoradiography. X-ray films were scanned with a BioRad densitometer and the area under the curve computed. Each group consisted of 10-12 rats. Mean + SEM of the densitometric units were calculated and arc shown as bar graphs. Results were analyzed by one-way analysis of variance. Uniform loading and integrity of RNA was ascertained by ethidium bromide staining of the ribo-
somal bands. The same blots were subsequently probed with a 1.2 kilo basepair bovine @-actin (Degen et al., 1983) probe and quantified as above. Serum
17@-estradiol
1er.el.y
After clotting, serum was recovered by centrifugation for 15 min at 3000 x g. Duplicate determinations of Ez concentrations in trunk blood were by modification of a validated radioimmunoassay (Leung et al., 1978). E, extraction from serum (400 ~1) by ethyl ether (3 ml) was 91 c/c efficient. First antiserum ( 1 : 600,000) bound 58% of [jH]E, (140 Ci/mmol diluted to 15,000 cpm/ 100 ~1, Amersham, Oakville, Ont., Canada). Assay sensitivity was 6 pg/ml serum (t-test, P < 0.05). Assay mid-range was 200 pg/ml. Analysis was in one assay. Intra-assay coefficients of variation for charcoal stripped serum with added E2 were 11.0% (mean = 15 pg/ml) and 6.0% (mean = 50 pg/ml). Results Results of densitometric quantification of CaBP-Yk and p-actin mRNA in rat uterus during the estrous cycle are shown in Fig. 1. When 20 Fg of total RNA were used for CaBP-9k quantification an intense hybridization signal was obtained after overnight autoradiography with RNA from rats in proestrus (Fig. 28). These levels dropped about IO-fold by estrus. In the uterus of rats in diestrus. CaBP-9k mRNA levels fell below the detection limit in 12 rats and a trace was dctectable in four rats. The same RNA blot was subsequently probed with a bovine p-actin probe. A slight increase of p-actin was seen at the transition from diestrus to proestrus, which carried on into estrus. When the p-actin levels at diestrus were compared with levels at proestrus and estrus the 1.3- and 1.6-fold induction was found not significant. In Fig. 2, a representative Northern blot of 20 pug total RNA is shown. UV spectrophotometry at 260 nm and ethidium bromide staining were used to assure uniform sample loading (Fig. 2A). Hybridization of the blotted RNA demonstrated intense regulation of CaBP-9k (Fig. 2 B) and near consistent expression of p-actin (Fig. 2C).
UTERUS
(A)
18S-
Calblndin
AC,,”
DIESTRUS
C.lbl”dl”
AC,,”
PROESTR”S
C.lbl”d,”
AC,,” ESTRUS
Fig. 1. Results of densitometric quantification of CaBP-Yk and /I-actin mRNA in the rat uterus during the estrous cycle are shown. 20 fig of total RNA were subjected to Northern blot analysis. The blots were sequentially hybridized to a rat CaBP-9k and a bovine p-actin cDNA probe. Following autoradiography, X-ray films were scanned and area under the curve computed. Each group consisted of 9-16 rats. The mean+SEM are indicated in densitometric units. ** P < 0.0001 CaBP-9k mRNA as compared to levels at proestrus.
The serum E2 concentrations are depicted in Fig. 3. Serum E, peaked during proestrus. The peak fell from 70 f 3.5 to 20 &-0.6 pg/ml at estrus. E, at diestrus was 43 + 6.2 pg/ml. Fig. 4 illustrates serum E, and CaBP-9k mRNA levels as the rats progress through the estrous cycle. The values from Figs. 1 and 2 were combined to illustrate temporal relationships between changing hormone levels and CaBP-9k mRNA expression.
(C)
p-actin-
Fig. 2. A: Ethidium bromide stained RNA gel of three representative samples out of each group tproestrus. estrus and diestrus). Samples were treated as described in Fig. 1. B: Autoradiograph of the RNA blot probed with CaBP-Ok. C: The same blot was subsequently probed with the p-actin probe. P. proestrus; E, estrus; D, diestrus.
dent. Based on previous findings in vivo and in vitro (Darwish et al., 1990; L’Horset et al., 1990), an effect was expected due to the estrogen con-
"1
Discussion The present study was carried out to determine any effects of hormonal changes during the estrous cycle on the CaBP-9k gene expression. For determination of the stage of the animals within the estrous cycle we used standard procedure of microscopic and macroscopic examination of the reproductive organs. Animals with inconclusive characteristics were excluded from the study. Serum E, was measured to correlate hormone levels with CaBP-9k mRNA gene expression. A rather rigorous regulation of CaBP-9k mRNA levels during the estrous cycle was evi-
Dle*lr”S
Ploestrus
Es,,“*
Fig. 3. Serum 17@-estradiol levels are shown as determined by radioimmunoassay in trunk blood of rats sacrificed on the morning (1O:OO a.m.) of diestrus, proestrus and estrus. Each group consisted of 9-16 animals. The mean? SEM are indi-
122
.
.
Fig. 4. The data from Figs. 1 and 3 are combined to illustrate the correlation of 17P-estradiol and CaBP-9k mRNA levels. There was no direct correlation of hormone levels and CaBPYk expression. The temporal patterns indicate a rise in hormone levels preceding the induction of CaBP-Yk mRNA. C’aBP-Yk mRNA is still detectable following the decline of 17p-estradiol from proestrus to estrus. Despite rising 17prstradiol levels at diestrus CaBP-Yk mRNA is not detectable (E. estrus; D, diestrus: P. proestrus).
trol of CaBP-9k gene in the rat uterus. Therefore, high expression of CaBP-9k gene was expected during proestrus when E, levels are elevated. Only 24 h later at estrus CaBP-9k mRNA levels had decreased sharply by 90% and fell below the detection limit at diestrus in 12 out of 16 rats. Despite 2-fold higher Ez levels at diestrus compared to estrus, CaBP-9k mRNA levels were higher at estrus. Fig. 4 illustrates the temporal relationship between changes in E, levels and CaBP-9k mRNA expression. From these data, it appears that a lag period occurs at diestrus, in which rising E, levels have not yet induced CaBP-Yk gene expression. Another possible explanation for these dynamics could be that a threshold level of E, combined with decreased serum progesterone is necessary for stimulation of CaBP-Yk gene transcription. The time course of CaBP-9k mRNA induction following a single injection of Ez into ovariectomized rats has been studied (L’Horset et al., 1990). A significant induction occurred at 6 h with a maximum at 12 h post-injection. After 48 h the mRNA levels decreased sharply. These results are not in agreement with data by Darwish et al. (1990>, where a single dose of E, caused persistent induction at 26 and 50 h post-injection. The dose used by L’Horset et al. (1990) was lo-fold higher (10
pg/kg) than used by Darwish et al. (1990). This discrepancy is of particular interest, when the results are compared to the present study. In intact animals in this study, it appeared that the high expression of CaBP-9k mRNA at proestrus is rapidly down-regulated within 24 h (estrus). After 4X h post-proestrus, steady-state CaBP-9k mRNA levels are below the detection limit. This implies that a sharp decrease of transcription rate combined with a rapid degradation of the RNA is caused by the physiological, hormonal changes in estrus and diestrus. Effects of E, on both the rate of gene transcription and RNA stability have been described for other genes, such as the vitellogenin in Xenopus liver (Brock and Shapiro, 1983). In this regard L’Horset et al. (1990) detected two mRNA species of CaBP-9k in rat uterus, about 50 nucleotides different in length. In vitro translation of the mRNA did not reveal synthesis of different CaBP-9k proteins. The authors suggested possible variation in the length of the polyA tail or the 5’ and 3’ non-coding regions of the transcripts. Two transcripts were not detected in the present study but there appears to be a difference in the molecular weight of the transcripts at proestrus compared to estrus (Fig. 2B). The low resolution of the 1% agarose gels used for the present experiments may prevent detection of two transcripts of similar size in one sample. The apparent heterogeneity of the CaBP9k mRNA warrants investigation of structural differences between the two transcripts. In conclusion, this study demonstrated a tight regulation of uterine CaBP-9k mRNA levels during the estrous cycle. The regulation is likely directed by changes in E, and progesterone and takes place at transcriptional and post-transcriptional levels. It will be important to measure CaBP-9k protein concentrations under the same conditions and to correlate changes in mRNA levels with actual protein levels.
References Brehier, A. and Thomasset, M. (1990) Endocrinology 127, X-X-587. Brock, M.L. and Shapiro, D.J. (1983) Cell 34, 2077214. Bruns, M.E., Overpeck, J.G., Smith, G.C., Hirsch, G.N., Mills, S.E. and Bruns, D.E. (19X8) Endocrinology 122.2371-2378.
123 Christakos, S., Gabrielides, C. and Rhoten, W.B. (1989) Endoer. Rev. 10, 3-26. Chymoczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162, 156-15’). Dar-wish, H., Krisinger. J., Strom, M. and DeLuca, H.F. (1987) Proc. Natl. Acad. Sci. USA 84, 6108-6111. Darwish, H., Krisinger. J., Furlow, J.D., Smith, C., Murdoch, F.E. and DeLuca, H.F. (1990) J. Biol. Chem. 266, 551-558. Degen, J.. Neubauer, M.. Freizner Degen, S.J., Seyfried, C.E. and Morris, D. (1983) J. Biol. Chem. 258. 12153-12162. Delorme, A.C., Danan, J.L., Acker. M.G., Ripoche. M.A. and Mathieu, H. (1983) Endocrinology 113. 1340-1347. Glisin. V., Crkvenjakov, R. and Byus, C. (1974) Biochemistry 13, 263332637. Kessler, M.A. and DeLuca, H.F. (1985) Arch. Biochem. Biophys. 236, 17-25. Krisinger, J.. Strom, M., Darwish. H.M., Perlman, K.. Smith, C. and DeLuca, H.F. (1990) J. Biol. Chem. 266. 1910-1913.
Kyozuka, M., Crankshaw, J., Berezin, I., Collins, S. and Daniel, E. (1987) Can. J. Physiol. Pharmacol. 65, 1966-1975. Leung, P.C.K., Goldenberg, S.R. and Armstrong, D.T. (1978) Biol. Reprod. 19, 1036-1039. L’Horset, F., Perret, C., Brehier, A. and Thomasset, M. (1990) Endocrinology 127, 2X91-2897. Mathieu. C., Mills, S., Burnett, S., Cloney, D., Bruns, D. and Bruns. M.E. (1989) Endocrinology 125, 2745-2750. Roche, C., Bellaton, C., Pansu, D., Miller, A. and Bronner, F. (1986) Am. J. Physiol. 251, G314-G320. Savineau, J.. Mironneau, J. and Mironneau. C. (1988) Pfliig. Arch. Eur. J. Physiol. 411. 296-303. Warembourg, M., Perret, C. and Thomasset, M. (1987) Cell Tissue Res. 247, 51-57. Wasserman. R.H. and Fullmer. C.S. (1989) Adv. Exp. Med. Biol. 24Y, 45-65.
MOLCEL
119
02786
Calbindin-D,,
J. Krisinger,
mRNA is tightly regulated in the rat uterus J.L. Dann, W.D. Currie,
during the estrous cycle
E.B. Jeung and P.C.K. Leung
Deparrmrnt of Ohstrtrics and Gynaecology, Unirmity of’ British C‘olmha.
Key words: Calbindin;
Uterus;
Estrogen;
(Received
27 February
Estrous
cycle: Actin
1992; accepted
3 April
Vancoulscv, B.C. VSZ 4H4. Canada 1992)
Summary Calbindin-D,, (CaBP-9k) is a cytosolic calcium binding protein with a molecular weight of 9000. CaBP-9k is mainly expressed in intestine, uterus and placenta, with intestinal levels controlled by vitamin D and uterine levels controlled by estrogens. CaBP-9k mRNA levels were measured in rat uterus throughout the estrous cycle. On the morning of proestrus, estrus and diestrus animals were sacrificed. Serum 17/3-estradiol concentrations were determined using a radioimmunoassay. Whole uterus was used for preparation of total RNA. Northern blot analysis was performed to quantify CaBP-9k and p-actin mRNA. CaBP-9k levels were highest at proestrus, dropped lo-fold at estrus and were not detectable at diestrus. @Actin levels did not change significantly throughout the estrous cycle. Peak 17&estradiol concentrations coincided with maximum CaBP-9k mRNA expression at proestrus. Despite minimal concentrations of 17/3-estradiol at estrus, CaBP-9k mRNA was still present at 10% of the proestrus level. At diestrus, CaBP-9k mRNA was not detectable despite increasing 17P-estradiol. It is concluded that CaBP-9k is subject to 17j3-estradiol regulation during the estrous cycle. Correlation between CaBP-9k mRNA and 17P-estradiol levels indicates a lag period for CaBP-9k induction in diestrus following a rise in steroid hormone levels.
Introduction Calbindin-D,, (CaBP-9k) is a 9000 M, cytosolit calcium binding protein expressed predominantly in duodenum, placenta and uterus (Christakos et al., 1989). In the duodenum, it is transcriptionally controlled by 1,25dihydroxy-
Correspondence to: Dr. J. Krisinger. The Research Centre, Dept. of OB/GYN, No. 178 - 950 W. 28th Ave., Vancouver, B.C. V5Z 4H4, Canada. Tel. (604) 8752433; Fax 875.2496. This work was supported by grants from the British Columbia Health Research Foundation No. 7 (90-2). The Molly Towell Perinatal Research Foundation. and the Medical Research Council of Canada (No. MT771 1).
vitamin D,, the hormonal form of vitamin D (Kessler and DeLuca, 198.5; Brehier and Thomasset, 1990; Krisinger et al., 1990) and is involved in intestinal calcium absorption (Roche et al., 1986; Wasserman et al., 1989). The function of uterine CaBP-Ok remains unknown. It has been postulated that the protein is involved in controlling the intra-myometrial calcium concentration via estrogen-mediated hormonal control (Bruns et al., 1988). By virtue of its calcium binding activity, CaBP-9k may decrease intracellular free calcium concentration or act as a calcium controlled mediator of uterine activity similar to calmodulin (Kyozuka et al., 1987; Savineau et al., 1988). The uterine CaBP-9k is tissue specifically controlled
120
by 17@-estradiol (Ez) at the transcriptional level mediated by an estrogen response element near the CaBP-9k gent promoter (Darwish et al., 1990). Histochcmical studies have shown that CaBP-9k is cxprcsscd in myometrium and endometrium (Dclorme et al., lYX3; Warembourg et al., 1987; Bruns et al., 198X; Mathieu et al., lYX9). After ovariectomy, uterine mRNA encoding CaBP-9k falls below detection levels and expression can be restored by E2 administration (Darwish et al., lY90; L’Horset et al., 1990). The present study examined the possible influence of the estrous cycle on the expression pattern of CaBP-Yk in the ‘uterus. Material and methods Animals
Two- to three-month female Sprague-Dawley rats were fed rat chow and maintained under a 12 h light/dark cycle. On the morning of the experiment (1O:OO a.m.), a vaginal smear was performed and rats were sacrificed by decapitation after halothane/N,O anesthesia. Blood was collected and the abdominal cavity opened by a midventral incision. Phase of the estrous cycle was immediatcly determined by vaginal exfoliate cytology and confirmed by dissecting microscopic examination of ovarian ultrastructures and the macroscopic appearance of the uterus. Quantifi;catiotz of CaBP-9k
mRNA
Whole uteri were homogenized and total RNA prepared by the guanidinium/CsCl procedure (Glisin et al.. 1974; Chomczynski and Sacchi, lYX7). Standard Northern analysis was performed with 20 pg of total RNA. A 295 nucleotide rat cDNA probe (Darwish ct al., 1987) was synthesized by random primed synthesis using [cu” P]dCTP. After stringent hybridization and washing, the blots were subjected to autoradiography. X-ray films were scanned with a BioRad densitometer and the area under the curve computed. Each group consisted of 10-12 rats. Mean + SEM of the densitometric units were calculated and arc shown as bar graphs. Results were analyzed by one-way analysis of variance. Uniform loading and integrity of RNA was ascertained by ethidium bromide staining of the ribo-
somal bands. The same blots were subsequently probed with a 1.2 kilo basepair bovine @-actin (Degen et al., 1983) probe and quantified as above. Serum
17@-estradiol
1er.el.y
After clotting, serum was recovered by centrifugation for 15 min at 3000 x g. Duplicate determinations of Ez concentrations in trunk blood were by modification of a validated radioimmunoassay (Leung et al., 1978). E, extraction from serum (400 ~1) by ethyl ether (3 ml) was 91 c/c efficient. First antiserum ( 1 : 600,000) bound 58% of [jH]E, (140 Ci/mmol diluted to 15,000 cpm/ 100 ~1, Amersham, Oakville, Ont., Canada). Assay sensitivity was 6 pg/ml serum (t-test, P < 0.05). Assay mid-range was 200 pg/ml. Analysis was in one assay. Intra-assay coefficients of variation for charcoal stripped serum with added E2 were 11.0% (mean = 15 pg/ml) and 6.0% (mean = 50 pg/ml). Results Results of densitometric quantification of CaBP-Yk and p-actin mRNA in rat uterus during the estrous cycle are shown in Fig. 1. When 20 Fg of total RNA were used for CaBP-9k quantification an intense hybridization signal was obtained after overnight autoradiography with RNA from rats in proestrus (Fig. 28). These levels dropped about IO-fold by estrus. In the uterus of rats in diestrus. CaBP-9k mRNA levels fell below the detection limit in 12 rats and a trace was dctectable in four rats. The same RNA blot was subsequently probed with a bovine p-actin probe. A slight increase of p-actin was seen at the transition from diestrus to proestrus, which carried on into estrus. When the p-actin levels at diestrus were compared with levels at proestrus and estrus the 1.3- and 1.6-fold induction was found not significant. In Fig. 2, a representative Northern blot of 20 pug total RNA is shown. UV spectrophotometry at 260 nm and ethidium bromide staining were used to assure uniform sample loading (Fig. 2A). Hybridization of the blotted RNA demonstrated intense regulation of CaBP-9k (Fig. 2 B) and near consistent expression of p-actin (Fig. 2C).
UTERUS
(A)
18S-
Calblndin
AC,,”
DIESTRUS
C.lbl”dl”
AC,,”
PROESTR”S
C.lbl”d,”
AC,,” ESTRUS
Fig. 1. Results of densitometric quantification of CaBP-Yk and /I-actin mRNA in the rat uterus during the estrous cycle are shown. 20 fig of total RNA were subjected to Northern blot analysis. The blots were sequentially hybridized to a rat CaBP-9k and a bovine p-actin cDNA probe. Following autoradiography, X-ray films were scanned and area under the curve computed. Each group consisted of 9-16 rats. The mean+SEM are indicated in densitometric units. ** P < 0.0001 CaBP-9k mRNA as compared to levels at proestrus.
The serum E2 concentrations are depicted in Fig. 3. Serum E, peaked during proestrus. The peak fell from 70 f 3.5 to 20 &-0.6 pg/ml at estrus. E, at diestrus was 43 + 6.2 pg/ml. Fig. 4 illustrates serum E, and CaBP-9k mRNA levels as the rats progress through the estrous cycle. The values from Figs. 1 and 2 were combined to illustrate temporal relationships between changing hormone levels and CaBP-9k mRNA expression.
(C)
p-actin-
Fig. 2. A: Ethidium bromide stained RNA gel of three representative samples out of each group tproestrus. estrus and diestrus). Samples were treated as described in Fig. 1. B: Autoradiograph of the RNA blot probed with CaBP-Ok. C: The same blot was subsequently probed with the p-actin probe. P. proestrus; E, estrus; D, diestrus.
dent. Based on previous findings in vivo and in vitro (Darwish et al., 1990; L’Horset et al., 1990), an effect was expected due to the estrogen con-
"1
Discussion The present study was carried out to determine any effects of hormonal changes during the estrous cycle on the CaBP-9k gene expression. For determination of the stage of the animals within the estrous cycle we used standard procedure of microscopic and macroscopic examination of the reproductive organs. Animals with inconclusive characteristics were excluded from the study. Serum E, was measured to correlate hormone levels with CaBP-9k mRNA gene expression. A rather rigorous regulation of CaBP-9k mRNA levels during the estrous cycle was evi-
Dle*lr”S
Ploestrus
Es,,“*
Fig. 3. Serum 17@-estradiol levels are shown as determined by radioimmunoassay in trunk blood of rats sacrificed on the morning (1O:OO a.m.) of diestrus, proestrus and estrus. Each group consisted of 9-16 animals. The mean? SEM are indi-
122
.
.
Fig. 4. The data from Figs. 1 and 3 are combined to illustrate the correlation of 17P-estradiol and CaBP-9k mRNA levels. There was no direct correlation of hormone levels and CaBPYk expression. The temporal patterns indicate a rise in hormone levels preceding the induction of CaBP-Yk mRNA. C’aBP-Yk mRNA is still detectable following the decline of 17p-estradiol from proestrus to estrus. Despite rising 17prstradiol levels at diestrus CaBP-Yk mRNA is not detectable (E. estrus; D, diestrus: P. proestrus).
trol of CaBP-9k gene in the rat uterus. Therefore, high expression of CaBP-9k gene was expected during proestrus when E, levels are elevated. Only 24 h later at estrus CaBP-9k mRNA levels had decreased sharply by 90% and fell below the detection limit at diestrus in 12 out of 16 rats. Despite 2-fold higher Ez levels at diestrus compared to estrus, CaBP-9k mRNA levels were higher at estrus. Fig. 4 illustrates the temporal relationship between changes in E, levels and CaBP-9k mRNA expression. From these data, it appears that a lag period occurs at diestrus, in which rising E, levels have not yet induced CaBP-Yk gene expression. Another possible explanation for these dynamics could be that a threshold level of E, combined with decreased serum progesterone is necessary for stimulation of CaBP-Yk gene transcription. The time course of CaBP-9k mRNA induction following a single injection of Ez into ovariectomized rats has been studied (L’Horset et al., 1990). A significant induction occurred at 6 h with a maximum at 12 h post-injection. After 48 h the mRNA levels decreased sharply. These results are not in agreement with data by Darwish et al. (1990>, where a single dose of E, caused persistent induction at 26 and 50 h post-injection. The dose used by L’Horset et al. (1990) was lo-fold higher (10
pg/kg) than used by Darwish et al. (1990). This discrepancy is of particular interest, when the results are compared to the present study. In intact animals in this study, it appeared that the high expression of CaBP-9k mRNA at proestrus is rapidly down-regulated within 24 h (estrus). After 4X h post-proestrus, steady-state CaBP-9k mRNA levels are below the detection limit. This implies that a sharp decrease of transcription rate combined with a rapid degradation of the RNA is caused by the physiological, hormonal changes in estrus and diestrus. Effects of E, on both the rate of gene transcription and RNA stability have been described for other genes, such as the vitellogenin in Xenopus liver (Brock and Shapiro, 1983). In this regard L’Horset et al. (1990) detected two mRNA species of CaBP-9k in rat uterus, about 50 nucleotides different in length. In vitro translation of the mRNA did not reveal synthesis of different CaBP-9k proteins. The authors suggested possible variation in the length of the polyA tail or the 5’ and 3’ non-coding regions of the transcripts. Two transcripts were not detected in the present study but there appears to be a difference in the molecular weight of the transcripts at proestrus compared to estrus (Fig. 2B). The low resolution of the 1% agarose gels used for the present experiments may prevent detection of two transcripts of similar size in one sample. The apparent heterogeneity of the CaBP9k mRNA warrants investigation of structural differences between the two transcripts. In conclusion, this study demonstrated a tight regulation of uterine CaBP-9k mRNA levels during the estrous cycle. The regulation is likely directed by changes in E, and progesterone and takes place at transcriptional and post-transcriptional levels. It will be important to measure CaBP-9k protein concentrations under the same conditions and to correlate changes in mRNA levels with actual protein levels.
References Brehier, A. and Thomasset, M. (1990) Endocrinology 127, X-X-587. Brock, M.L. and Shapiro, D.J. (1983) Cell 34, 2077214. Bruns, M.E., Overpeck, J.G., Smith, G.C., Hirsch, G.N., Mills, S.E. and Bruns, D.E. (19X8) Endocrinology 122.2371-2378.
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