Department of Molecular and Cell Biology, Baylor College of Medicine (E.M.A., B.W.O.), Houston, Texas 77030; Department of Pharmacology, University of Louisville (D.Z.), Louisville, Kentucky 40292; Krieger School of Arts and Sciences, The John Hopkins University (M.R.), Baltimore, Maryland 21218; and Department of Cell Biology and Physiology, University of Pittsburgh (S.O.), Pittsburgh, Pennsylvania 15261
Address all correspondence and requests for reprints to: Dr. Ede Marie Apostolakis, Department of Molecular and Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: edea{at}bcm.tmc.edu.
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ABSTRACT |
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INTRODUCTION |
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As sex steroids and their cognate receptors [estrogen receptor (ER
) and progesterone receptor (PR)] exert profound influence on the developing and adult brain, it is critical to fully understand the molecular regulation of steroid receptors by coactivator complexes in the intact animal. Little is known about the specific distribution pattern of SRCs in the female brain. Coactivator genes have been detected by in situ hybridization and Northern blot analysis in rodent hypothalamii (4, 5).
The ovarian hormones estrogen (E) and progesterone (P) regulate cellular functions in the brain that exquisitely control sexual behavior (6). Priming with E for 48 h increases the synthesis of PR in the female hypothalamic ventromedial nucleus (VMN) (7), an event that coincides with the ability of P to facilitate PR-dependent sexual receptivity (8, 9). In addition, we have reported the existence of cross-talk among steroid hormone-, neurotransmitter-, and growth factor-initiated pathways for sexual receptivity in female rats and mice (10, 11, 12, 13). Because SRC-1, SRC-2, and SRC-3 alter the efficiency of steroid receptors for E and P in vitro, we examined whether these coactivators are required for steroid receptor function in intact biological systems. To this end, we used the rat and mouse reproductive behavioral models to determine whether there is a biological relationship between steroid receptors and their coactivators. We provide evidence for the requirement of hypothalamic SRC-1 and SRC-2, but not SRC-3, for steroid-dependent sexual behavior in E-primed females. We clarify the biological mechanism of SRC activity by showing that SRC-1- and SRC-2-AS suppress both the synthesis of E-dependent PR in the VMN and ER-dependent sexual behavior. Next, we demonstrate that the biological role of hypothalamic steroid receptors is regulated by the distribution of SRCs in the female VMN. Finally, we provide evidence that SRC-2 is a critical component of the molecular mechanism for developmental adaptation in the genetic absence of SRC-1, thus unmasking a role for SRC-2 in the development of the female VMN. The present findings delineate several mechanisms within the female hypothalamus and substantiate the requirement for gene transcription in this steroid-induced behavioral response.
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RESULTS |
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As expected, sexual behavior was not exhibited by control treated rats administered vehicle, E, or P only in each experiment (Fig. 2, AC, and Fig. 3A
, bars 13); however, receptivity was displayed after E plus P compared with vehicle, E, or P only results (bars 4; by two-way ANOVA, P < 0.001). Regardless of type of SRC oligonucleotide, both rSRC-RS and rSRC-RS plus E had no effect on behavior in each experiment (Fig. 2
, AC, and Fig. 3A
, bars 56; P > 0.05), whereas sexual behavior was exhibited by rSRC-RS, E, plus P (bars 7; P < 0.001). Likewise, compared with E plus P, those animals treated with rSRC-RS and E plus P had comparable levels of receptive behavior in each experiment (Fig. 2
, AC, and Fig. 3A
, bars 4 vs. 7; P > 0.05). Also, neither rSRC-1-AS nor rSRC-1-AS plus E had an effect on behavior compared with vehicle, E, or P only (Fig. 2
, AC, and Fig. 3A
, bars 89; P > 0.05). These data from control conditions in each behavioral study confirm the absence of any nonspecific effect of steroid and/or oligonucleotide treatment on behavior.
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rSRC-2-AS Also Inhibits P Induction of Sexual Behavior
Significantly, 2 nM rSRC-2-AS inhibited P-facilitated receptivity in E-primed females compared with negative control treatment groups (P < 0.05; Fig. 2B, bar 10), demonstrating that a second coactivator in the VMN is required for steroid-dependent behavior. The optimal oligonucleotide concentration was about 2 nM, icv, again implicating a threshold level for the oligonucleotide effect. Hence, both SRC-1 and SRC-2 (also known as TIF2/GRIP1) are required for functional activity of steroid receptors in the VMN and subsequent steroid-dependent behavior.
P Facilitates Receptivity Despite Treatment with rSRC-3-AS
Next, we addressed the question of whether a third related coregulator, rSRC-3, plays a role in regulating steroid-dependent behavior. Interestingly, P facilitated lordosis in the experimental E-primed females pretreated with rSRC-3-AS (Fig. 2C, bar 10); hence, there was no significant difference between the E plus P treatment group and the E, rSRC-3-AS, plus P treatment group (P > 0.05). The data suggest that SRC-3 (also called p/CIP) is not required for this steroid receptor activity in the female hypothalamus.
rSRC-1-, rSRC-2-, and rSRC-3-ASs Suppress Protein Expression of irSRCs
Western blot analysis detected irSRC-1 and SRC-2 proteins in the medial basal hypothalamus (MBH) of both wild-type mice (Fig. 2D, left and middle panels, respectively) regardless of E and random sequence oligonucleotides. SRC-1-AS reduced its target protein expression in the MBH (Fig. 2D
, left panels). Likewise, SRC-2-AS had a suppressive effect on VMN SRC-2 protein expression (Fig. 2D
, middle panels). To more easily demonstrate the effect on protein levels, SRC-3 oligonucleotides were injected into the hippocampus, where expression was highest in the brain. In contrast, SRC-3 was expressed at very low levels in the MBH (Fig. 2D
, right panels). As expected, SRC-3 protein was reduced by SRC-3-AS (Fig. 2D
, right panels). As in mice, SRC-ASs designed for use in rats also suppressed their target proteins in rat MBH and hippocampus (data not shown). Thus, the SRC oligonucleotides were specific for their respective target proteins.
Interruption of E-Dependent Cascade Disrupts Reproductive Behavior
It has been proposed that initial ER activation by E must be followed by hormone stimulation approximately 24 h later to exert an effect on the protein cascade that results in sexual behavior (6). As a control for the late effect of disrupting ER
, oligonucleotides to ER
were given 24 h before P (Fig. 3A
, closed bar). It is noteworthy that ER
-AS given 24 h after E blocked P-induced lordosis in a manner similar to that of concurrent administration (open bar). This finding demonstrates that the transcriptional efficiency of ER
is also critical during the second half of the cascade that accounts for reproductive behavior. Further, sexual behavior was displayed in a dose-dependent manner with varying concentrations of AS regardless of the time of oligonucleotide administration (04 nM; data not shown), suggesting that a minimal threshold level of ER
-dependent transcription is needed for subsequent behavioral changes. Together with the above behavioral results for SRC-AS treatment, the data suggest that SRC-1 and SRC-2 are also essential for late ER
activation and for the likely protein cascade that results in sexual behavior.
ASs to rSRC-1 and rSRC-2, But Not rSRC-3, Block ER-Induced PR in MBH and VMN
Compared with PR content in untreated MBH (Fig. 3B, bar 1), E priming enhanced PR content in MBH (bar 2; P < 0.05). As expected, rRS treatment had little effect either alone (Fig. 3B
, bars 35) or with E priming (bars 68). Also, the MBH PR contents in females treated only with rSRC-1-AS (Fig. 3B
, bar 9), rSRC-2-AS (bar 10), or rSRC-3-AS (bar 11) were comparable with those in untreated females (P > 0.05). Of interest, both rSRC-1-AS and rSRC-2-AS attenuated E-induced synthesis of PR (Fig. 3B
, bars 1213; P < 0.05), consistent with the absence of ER
-driven transcription. rSRC-3-AS failed to disrupt E-induced PR in the MBH (Fig. 3B
, bar 14; P > 0.05), suggesting that rSRC-3 is not required for ER
-dependent transcription in the MBH. Taken together, these findings depict the in vivo regulatory effect of SRC-1 and SRC-2 on targeted ER
transcription in the MBH.
Immunoreactive PR Are Not Detected in the Hypothalamic VMN after Pretreatment with rSRC-1- and rSRC-2-AS
Immunoreactive PR were detected in the hypothalamic VMN of E-primed females receiving icv rSRC-1-RS (Fig. 3C, upper left panel), rSRC-2-RS (upper middle panel), and rSRC-3-RS (upper right panel), but not rSRC-1- or rSRC-2-AS (lower left and lower middle panels, respectively). Notably, rSRC-3-AS failed to inhibit the induction of irPR in the VMN of females administered E (Fig. 3C
, lower right panel). In the absence of E, oligonucleotides had no apparent effect (insets for each panel). As expected, no immunoreactivity was noted in tissue stained only with primary antibody or secondary antibody (data not shown). Together, the data provide evidence for the disruption of E-induced PR synthesis after rSRC-1- and rSRC-2-AS, but not rSRC-3-AS, in the VMN.
rSRC-1- and rSRC-2-AS Inhibit the Facilitation of ER-Dependent Behavior
To test the function of hypothalamic ER, we administered high dose E, which is known to induce behavior independent of P and PR (13). Estradiol (200 µg, sc) was given concurrently with oligonucleotides, and females were tested 44 h later. In control tests no receptivity was observed with vehicle (Fig. 3D
, bars 13) or the standard E-priming dose (bars 46); however, sexual behavior was displayed with high E (Fig. 3D
, bars 79), but not rRS only (bars 1012). With high E plus rSRC-1-RS (Fig. 3D
, open bar 13), rSRC-2-RS (closed bar 14), and rSRC-3-RS (hatched bar 15), receptivity was exhibited. As expected, rAS only controls failed to induce receptivity (Fig. 3D
, bars 1618). In the experimental animals treated with high E, both rSRC-1-AS and rSRC-2-AS blocked sexual behavior (Fig. 3D
, bars 1920), corroborating the hypothesis that both SRC-1 and SRC-2 are required for the in vivo function of hypothalamic ER
in intact females. Again, rSRC-3-AS had no effect on the behavioral effect of high E (Fig. 3D
, bar 21).
To further substantiate this, females were administered epidermal growth factor (EGF), an agent that activates ER in a ligand-independent manner for PR-independent receptivity (14). As shown in Fig. 3E
, where vehicle failed to alter behavior (Fig. 3E
, bars 13), EGF induced sexual behavior (Fig. 3E
, bar 46). Both rSRC-1-RS and rSRC-2-RS failed to alter behavior (Fig. 3E
, bars 79) and did not interfere with the behavioral effect of EGF in control tests (Fig. 3E
, bars 1012). None of the rSRC-ASs induced lordosis (Fig. 3E
, bars 1315), confirming the absence of a nonspecific oligonucleotide effect. Significantly, rSRC-1-AS and rSRC-2-AS (Fig. 3E
, bars 1617), but not rSRC-3-AS (bar 18), blocked EGF-facilitated receptivity. Together, the data substantiate the hypothesis that both SRC-1 and SRC-2 inhibit the functional activity of ER
in the intact animal.
Controls for AS Studies in Female SRC-1 Knockout Mice
Coactivator AS oligonucleotides were designed using to reported mouse sequences from the GenBank for all mouse studies. Again, oligonucleotides of the same length and base composition but randomized in sequence (RS) were used as controls for sequence-dependent and -independent interactions. None of the oligonucleotides used showed homology with other mouse genes, as reported in GenBank. The specificities of oligonucleotides are demonstrated in Fig. 2D.
Control doses of steroid in mice were 1 µg, sc, for E and 100 µg, sc, for P as described in Materials and Methods. For control treatments in both wild-type (open bars) and knockout (closed bars) SRC-1 mice in all experiments, neither vehicle, E, nor P only induced lordosis (Fig. 4, AC, bars 16, respectively). Wild-type SRC-1 mice exhibited a positive response with E plus P treatment compared with wild-type and knockout mice treated with vehicle, E, or P only (Fig. 4
, AC, bars 7; P < 0.001). Controls for RS treated failed to exhibit sexual behavior in all experiments with SC-1 wild type and their knockout counterparts (Fig. 4
, AC, bars 910) regardless of E priming (bars 1112). As expected, when P was given to those females, receptivity was displayed (Fig. 4
, A and C, bars 1314). For AS treatment, control treatments of AS (Fig. 4
, AC, bars 1516) alone (bars 17) and with (bars 18) E also were not associated with behavioral changes. Collectively, these findings confirm the absence of a nonspecific oligonucleotide effect on behavior in SRC-1 mice.
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mSRC-2-AS Blocks P-Facilitated Behavior in Both Wild-Type and Knockout SRC-1 Mice
Remarkably, AS to mSRC-2 inhibited sexual behavior in E- plus P-treated mice regardless of genotype (Fig. 4B, bars 1920) verifying that SRC-2 is an essential component of the molecular mechanism underlying steroid-induced reproductive behavior. Further, the ability of the SRC-2 AS to suppress lordosis in the SRC-1-null females indicates that SRC-2 overexpression is an important adaptation to the genetic loss of SRC-1. Collectively, the findings provide evidence that the sister protein mSRC-2 is required for steroid receptor-dependent behavior in SRC-1 mice regardless of genotype. Hence, mSRC-2 plays a critical adaptive role in the genetic absence of mSRC-1 in knockout mice.
Behavioral Studies in SRC-3 Knockout Mice
As described above for all tests in rats using mSRC-3 oligonucleotides, P facilitated sexual behavior in E-primed wild-type and knockout SRC-1 females administered mSRC-3-AS (Fig. 4C, bars 19 and 20; P > 0.05) compared with negative control treatment effects (bars 16, 912, and 1518). For comprehensiveness, SRC-3 knockout mice also were tested. Both wild-type and knockout E-primed female SRC-3 mice displayed P-facilitated lordosis (Fig. 4D
, bars 7 and 8), a steroid effect that was not altered by pretreatment with either SRC-3-RS (bars 13 and 14) or SRC-3-AS (bars 19 and 20). As expected, receptivity was not exhibited by females under negative control conditions (bars 16, 912, and 1518). Collectively, the mouse data provide additional evidence for the absence of functional mSRC-3 in the VMN as well as for the absence of cross-reactivity between SRC-3-AS and endogenous SRC-1 and SRC-2.
Hypothalamic SRC Distribution in Hypothalamic VMN
The sparse distribution of SRC-3 proteins and mRNA in the rodent MBH was confirmed by immunohistochemistry (IHC) and RT-PCR. Interestingly, irSRC-3 was seen in both the cytoplasm (Fig. 5A, n-arrow, brown staining) and nucleus of positive cells (Fig. 5A
, arrow and inset for representative tissue from a wild-type mouse). By RT-PCR, rSRC-3 mRNA was barely detectable in punched-out rat VMN (Fig. 5B
, right photomicrograph and histogram, V) regardless of E priming (E vs. vehicle treatment). In contrast, E priming appeared to enhance the relative expression of rSRC-2 mRNA in the VMN (Fig. 5B
, right photomicrograph and histogram). Collectively, the findings demonstrate the absence of sufficient distribution SRC-3 in the female rodent VMN and may explain the failure of SRC-3-AS to inhibit receptivity regardless of species, genotype, and steroid treatment. The data also support the possibility that SRC-2 mRNA expression may be regulated by E in the VMN. Finally, as icv SRC-3 AS treatment of both wild-type and knockout SRC-1 mice failed to inhibit steroid-dependent behavior and suppressed irSRC-3 cells in the hippocampus, there is no evidence of cross-reactivity of the SRC-3 AS with either SRC-1 or SRC-2.
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DISCUSSION |
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In concordance with previous in vitro reports on the ability of SRC-1 and SRC-2 to enhance ER- and PR-mediated gene transcription (14, 15), the current study demonstrates a strong correlation between the regional and relative expression of SRC proteins in the VMN and the display of steroid receptor-dependent behavior. SRC-1 was detected in the MBH of females by Western analysis (and in the VMN by IHC, our unpublished data), findings consistent with in situ hybridization for SRC-1 mRNA in the male rat brain (4). SRC-2 mRNA has been detected in whole male brain by blot analysis (16). In support of this finding, we found SRC-2 protein in the female MBH using Western blots (and in the VMN by IHC, our unpublished data). Although we detected SRC-2 mRNA by RT-PCR in punched-out female VMN by RT-PCR (and in a preliminary experiment using in situ hybridization and cDNA probes), an earlier study (4) failed to detect SRC-2 mRNA using in situ hybridization, suggesting that the expression of SRC-2 may be gender dependent. Moreover, gender differences have been noted for SRC-1 by Northern analysis (5). Alternately, methodological differences may explain the differences between studies, as oligonucleotide probes were used in the latter study (4).
ER-mediated induction of specific genes in the VMN is essential for sexual behavior (6). After 2440 h of E priming, the induction of PR mRNA and protein in the VMN allows for P-facilitated behavior within 1015 min after icv or intranuclear injection. Consequently, the biological mechanism by which diminished expression of SRC-1 and SRC-2 inhibits sexual behavior is of interest. As the effective timeframe for optimal SRC-1 and SRC-2 AS effect on behavior was 612 h after administration, the protein synthesis cascade induced after ER
occupancy would have been interrupted during the first 612 h in animals given E and AS concurrently. In the current study of E-primed females, PR protein content within the hypothalamus, specifically the VMN, was significantly reduced after treatment with AS to SRC-1 and to SRC-2, implicating an ER
-driven mechanism. Likewise, neither high dose E nor EGF alone facilitated sexual behavior when the females were pretreated with either of the two SRC ASs. These data are significant because high dose E- and EGF-induced receptivity are dependent on ER
and independent of PR (13). Collectively, the findings provide evidence that the biological mechanism by which SRC-1 and SRC-2 mediate receptivity is at least initially through the facilitation of ER
-dependent transcription.
Modulation of coactivator expression by hormones may represent another level of steroid receptor activity function in mammalians. In contrast to male hypothalamii (5), we found that irSRC-1 and irSRC-2 expression in the female VMN was maintained during E priming. Indeed, it is the possible that VMN SRC-2 mRNA expression is influenced by E. Thus, the data are consistent with the maintenance of the E-induced protein synthesis cascade that is required for approximately 48 h before female sexual receptivity is inducible (6). It would be interesting to know whether there are variable concentrations of different SRCs in the preoptic area as well as in the VMN across the estrous cycle in intact females. After all, gender- and time-specific changes in the relative amounts of coactivators and corepressors could explain the several phenomena in steroid biology, including the positive effect of E on the GnRH surge at the time of estrus and the refractive period for P 24 h after receptivity.
In the present study using antisense oligonucleotides we found no evidence for adaptive mechanisms to the acute loss of SRC-1 or SRC-2, e.g. both SRC-1 and SRC-2 were required for sexual behavior regardless of the species or genotype. Similarly, when SRC-1 AS was administered into the brain of pups early in postnatal life, ER-dependent sexual differentiation of the hypothalamus was disrupted (17). However, in our present study of mutant SRC-1 mice, null females displayed full feminization and a complete repertoire of proceptive and receptive behaviors comparable with their wild-type counterparts. Likewise, they were fertile, delivered on time, suckled normal litters of pups, and exhibited no phenotypic loss in size and growth (14). However, some loss was detected in those reproductive functions under multiple steroid receptor control, including uterine decidual response and elongation of ductal branches in the mammary gland, possibly indicating that some biological functions of the two coactivators may differ. Nevertheless, our findings in the brain suggest an adaptive mechanism during development for the absence of SRC-1. The likely mechanism is that the genetic absence of SRC-1 during development invokes up-regulation of SRC-2. Indeed, SRC-2 has been reported to be overexpressed in SRC-1-/- brain, but not mammary gland or uterus (14). Our results show that SRC-2 AS inhibited sexual behavior in both wild-type and knockout SRC-1 mice primed with E and challenged with P. Thus, SRC-2 serves an adaptive role in the genetic absence of SRC-1 to functionally compensate for the lack of a sister coactivator during development.
Clear evidence that SRC-1 and SRC-2 play fundamental roles in steroid receptor biology in the female brain has been presented, as SRC-1- and SRC-2-AS administered separately blocked the function and protein expression of the respective coactivators and synthesis of ER-dependent PR in the hypothalamus. Several lines of in vitro evidence argue that SRC-2 is interchangeable with SRC-1 (1, 3, 15, 18, 19, 20, 21, 22). In our studies we observed comparable levels of steroid-dependent receptivity (and no phenotypic characteristics) in both wild-type and heterozygous mice, indicating that the genetic loss of one gene copy has little if any biological effect on the adult female. Likewise, comparable doses (0.5, 1, and 2 nM) of SRC-1- and SRC-2-AS given individually had statistically similar effects on sexual behavior, and the optimal 2-nM dose of either AS suppressed MBH PR binding to statistically similar concentrations. Further animal studies are warranted to define the precise role that relative changes in SRC-1 and SRC-2 may play in physiology.
SRC-3 facilitates the efficiency of ER (and PR)-dependent transcription in cultured cells (1). Here, female rats primed with E exhibited full proceptive and receptive behaviors regardless of SRC-3-AS. In addition, SRC-3-AS did not block the synthesis of PR in the female rodent brain. This is consistent with the fact that little SRC-3 mRNA or immunoreactive protein is expressed in the female VMN. Further, SRC-3-/- mice displayed reproductive behavior despite the genetic absence of SRC-3. Collectively, the data suggest that SRC-3 is not required for the E-induced protein cascade in the VMN that results in reproductive behavior. These findings implicate the selective involvement of some, but not all, coactivators in specific steroid-driven functions of the brain. Moreover, we found no evidence of hormonal regulation of SRC-3 expression, and the genetic loss of SRC-1 failed to regulate SRC-3 expression in the female VMN. Taken together, we conclude that during development, the differential distribution of coactivators can be influenced by the absence of other coactivators and, in adult females, by the hormonal status. With the observations related to SRC-3, we now show that selective SRCs participate in equally selective biological processes that are mediated by steroid receptors.
In conclusion, the data demonstrate the exquisite nature and complexity of molecular regulation of steroid receptors by coactivators and delineate their relationship to basic reproductive physiology in mammalians. It is evident that the dual expression of SRC-1 and SRC-2 in the female VMN is essential for at least ER-dependent transcription. Our results also confirm the requirement for receptor-mediated transcription as a component of steroid-induced reproductive behavior and argue against a direct and sufficient membrane or nongenomic explanation for the response. Further, the intact animal appears to lack the ability to acclimate or adapt to acute short-term reduction of SRC-1 and SRC-2 levels. As the expression of SRC-2 in females is regulated by SRC-1 in the VMN during development, the data suggest that there is a critical window of time for such regulation. Finally, the distribution of SRC-3 in the brain is regionally discrete regardless of age, time, or absence of other coactivators. All in all, the putative ability of SRC-1 and SCR-2 to modulate the synthesis of E-dependent PR makes them participants in neuroendocrine function and regulators of this receptor-dependent behavior. These findings may have broad implications to development, normal steroid receptor biology, and disease states associated with steroid receptors and altered expression of SRCs.
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MATERIALS AND METHODS |
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Behavioral Testing and Stereotaxic Surgery
For each experiment a standard reproductive behavior paradigm for rats was used (6, 10, 11, 13). That is, 710 d after cannulation, E-primed (10 µg sc) females were screened for steroid-dependent behavior after P treatment (1 µg, icv, or 0.5 µg onto the VMN). Sexual behavior was measured within 15120 min after P challenge for lordosis quotient [(total number of positive responses divided by total number of mounts for a series of four males) ÷ 100] by an observer blind to individual animal treatment. For ER experiments, EGF (10 ng) was given icv 44 h after oligonucleotide pretreatment, and females were tested within 1 h for receptivity. Results are expressed as the percentage of positive responses for all females mounted by males ± SEM. Animals served as their own control for each experiment. Each experiment also included control groups of E- plus P-treated and non-E-primed females. Each group consisted of a minimum of six females, and each experiment was repeated two or three times.
In our mouse paradigm (13) females were ovariectomized, primed every 7 d for 4 consecutive wk with 1 µg E, sc, followed 40 h later by 100 µg P, sc, and tested in the male home cage with males of the same stain as the female. For ER experiments, behavior testing was performed 1560 min after EGF (0.1 ng, icv) treatment, and oligonucleotides were given 24 h before EGF treatment. The testing was performed in the dark under a red light and was continued until the male had mounted the experimental female 10 times. For experiments, females were cannulated in the third ventricle and recovered for 7 d. Each experiment consisted of a minimum of wild-type and mutant mice (n = 1216 for each genotype) under identical treatments. Animals served as their own control for each experiment. Each group consisted of a minimum of four females, and each experiment was repeated three or four times. The observer was blind to individual animal treatment and genotype for mice.
Cloning of SRC-1, SRC-2, and SRC-3 Genes Using RT-PCR
The cDNA coding for SRC genes was obtained using total RNA extracted from whole rat brain and probed by SuperScript RT-PCR (Life Technologies, Inc., Gaithersburg, MD). The PCR primers of SRC-1 were designed using human SRC-1 sequence (14) (accession codes U40396 and NM 003743). The sequence for the forward primer was ATG GAT CCA TGT AAT ACA AAC CC (amino acid 2341), and the reverse primer was CCA AAG CTT TGC TGC CTC ATA AGC (amino acid 3422). For cloning of SRC-2, a highly conserved region of hTIF2 (17) (accession code NM006540) and mouse GRIP sequence (24) (accession code U39060) was used for designing PCR primers (forward, GGA TCC AGT AAC TAT GCA CTG ATG, amino acid 448; reverse, AAG CTT GGG GGA TTC ATA TTA CC, amino acid 889). The PCR primers of SRC-3 were designed according to a conserved region of mouse p/CIP (18) (accession code AF000581) and human RAC-3 genes (25) (accession code AF010227). The primers were: forward (amino acid 368), GGA TCC TGA TGA TGA TGT TCA AAA AGC; and reverse (amino acid 768), AAG CTT GCA AAG CAC TGC ATT GTT TCA TAT C. The PCR products were cloned into pCR3.1 TA cloning vector (Invitrogen, San Diego, CA) and sequenced to confirm the identities of the coactivators.
Rodent SRC-1, SRC-2, and SRC-3 Oligonucleotides and Specificities
Cannulated rats were injected with 2 nM phosphorothioated oligonucleotides concurrently with E (10 µg sc). For rSRC-1 the sequence for AS was GCC ACT AAG GAA GGA TAC (sense strand, GTA TCC TTC CTT AGT GGC; amino acid 2240) and for RS the sequence was GTA TCC TTC CTT AGT GGC. The sequence for rSRC-2-AS was TG TGC TTC CTG GTT ATC AT (sense strand, AT GAT AAC CAG GAA GCA CA; amino acid 365384), and that for rSRC-2-RS was AAA TTA AAG GGG AAA CCC C. The sequence for SRC-3-AS was TG ATT ATC AAA CAC CAT TGA (sense strand, TCA ATG GTG TTT GAT AAT CA; amino acid 231250), and that for SRC-3-RS was ACG TAT ACA ACA ACT TAT TG. Preliminary experiments using fluorescent-labeled oligonucleotides that confirmed the presence of the oligonucleotides within VMN cells.
For mice, the mSRC-1-AS sequence was GCC ACT GAG GAA AGA CAC CA (26) (sense strand, TG GTG TCT TTC CTC AGT GGC; amino acid 34493468; accession code U64828), and the mSRC-1-RS sequence was GGG GGG TTT TTT TTA CCC CC. Mouse SRC-2 oligonucleotides were designed against the mGRIP (24) (accession code U64828) and a homologous portion of the hTIF2 (23) (accession code 008678, amino acid 15431560). For mSRC-2-AS the sequence was TTT GAG TGC ATA GTT ACT (amino acids 15841601; sense strand, AGT AAC TAT GCA CTC AAA), and for mSRC-2-RS the sequence was CAG ATA ATG ACT ACA TAC. Mouse SRC-3-AS was against the mouse p/CIP gene (18) (accession no. AF000581) with a sequence of AAA GCA CTG CAT TGT TTC (sense strand, GAA ACA ATG CAG TGC TTT; amino acids 776793), and the sequence for mSRC-3-RS was GC ATA CAA GAG CGT TTT A.
Western Analysis for Coactivators in Female MBH
Oligonucleotide specificity for rat and mouse SRCs was determined by Western blot analysis using MBH lysates prepared from rats and SRC-1 wild-type mice as described previously (17, 27). Cannulated ovariectomized females underwent sc E and icv oligonucleotide treatments as described above for behavioral studies. Based on the high abundance of detectable immunoreactive cells, SRC-3 specificity was tested using animals cannulated by stereotaxis in the hippocampus. All were deeply anesthetized 44 h after E treatment; MBH were excised, snap-frozen, and stored at 70 C. To optimize target protein expression, pools of three MBH per treatment group were homogenized in ice-cold lysis buffer, and supernatants were subjected to SDS-PAGE using 40 µg total protein in each lane of precast 816% Tris/glycerine gels. After transfer to polyvinylidene difluoride membrane, nonspecific binding was blocked by 5% nonfat dry milk in 0.1 M Tris-buffered saline/0.1% Tween (TBS-T) for SRC-1 and SRC-2 and 5% goat serum for SRC-3. Membranes were incubated with mouse monoclonal SRC-1 antibody (1:100) (28) in 2% nonfat dry milk in TBS-T, polyclonal SRC-2 antibody (clone M-20; 1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in TBS-T, or monoclonal SRC-3 antibody (1:400) (24) in TBS-T containing 1.5% goat serum and 3% BSA. SRC-3 antibody was provided by J. Torchia (The University of Western Ontario, London, Ontario, Canada). Twenty-four hours later, blots were visualized using horseradish peroxidase-linked secondary antibodies (goat antimouse for SRC-1, 1:5000; rabbit antigoat for SRC-2, 1:500; goat antirabbit for SRC-3; 1:1000; Vector Laboratories, Inc., Burlingame, CA), followed by chemiluminescence with an enhanced chemiluminescence kit (Pierce Chemical Co., Rockford, IL). Nonspecific bands appeared in all lanes for each protein and served as a control for the total amount of protein loaded. Levels of proteins were revealed by autoradiography and quantified by densitometry. The experiments were repeated twice.
IHC for irSRC Protein Expression in VMN
The presence of irSRC protein was confirmed in the VMN by IHC. Rodents were deeply anesthetized 44 h after E treatment and underwent transcardiac perfusion with heparinized PBS, followed by fixation with cold 4% paraformaldehyde in PBS for 1 h. Tissue was dehydrated in ethanol at graded concentrations (70100%) for 1 h each and clarified with xylene using an automated Citadel 2000 Wax Bath/Tissue Processor and Histrocentre 2 (Shandon Lipshaw, Pittsburgh, PA). The paraffin blocks were stored at 4 C until sectioning (57 µm using an AS325 Rotary Microtome, Shandon Lipshaw, Pittsburgh, PA). Mounted sections were deparaffinized with xylene, dehydrated in ethanol, and then rehydrated in PBS. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol, and tissue was treated with 1% Antigen Retrieval Solution (Vector Laboratories, Inc.). To diminish nonspecific binding, sections were incubated in 5% normal goat serum (Vector Laboratories, Inc.), followed by overnight incubation in the polyclonal goat anti-SRC antibodies (clone C-20 for SRC-1 and clone M-20 for SRC-2; 1:200; Santa Cruz Biotechnology, Inc.). SRC-3 antibody was provided by J. Torchia (18). After incubating with biotinylated second antibody, antigen was visualized using the Elite Vectastain kit (Vector Laboratories, Inc.), and sections were counterstained with hematoxylin. PBS was used to wash sections between all steps. To determine the specificity of each SRC antiserum, control sections were included in which only the primary or secondary antibody was used. Also, as a competitive control, some sections were incubated in 5x blocking peptide and primary antibody (1:200). To verify the specificity and absence of cross-reactivity in vivo, IHC was performed using knockout mice for SRC-1, SRC-2, and SRC-3.
PR Cytosolic Assay
Preparation of animals and cytosol.
Ovarectomized rats were treated with E (10 µg, sc) for 3 consecutive d and were decapitated on the next day. MBH were excised as described previously (29) and then homogenized by hand in freshly prepared ice-cold TEGT buffer, pH 7.4 [10 mM Tris-HCl, 10 mM sodium EDTA, 10% (vol/vol) glycerol, and 12.0 mM monothioglycerol]. Homogenized tissue was ultracentrifuged at 48,000 rpm for 30 min at 4 C.
LH-20 columns for gel filtration.
Sephadex LH-20 (Amersham Pharmacia Biotech, Piscataway, NJ) was expanded in TEGT buffer at 4 C, poured into 8 x 34-mm columns, and equilibrated overnight at 4 C. On the day of tissue collection, ultracentrifuged supernatant was added 30 min after LH-20 columns were primed with fresh TEGT buffer at 4 C.
PR cytosolic assay.
Supernatant (100 µg) was added to 1 nM [3H]R5020 (1 nM) to determine the total [3H]progesterone exchanged and 100 µg plus [3H]R5020 (0.4 nM) and unlabeled R5020 (100 nM) (250-fold excess). After 24-h incubation, bound and free [3H]R5020 were separated by gel filtration over Sephadex LH-20 columns. Samples were counted in scintillation liquid and compared with the standard curve for determination of concentrations. All samples were processed in duplicate for each animal within each treatment group. Each in vivo treatment group was composed of three MBH, and the study was replicated twice.
RT-PCR for SRCs in the Female VMN
Ovarectomized animals (n = 12) were divided into two groups, and one received E priming. Animals were decapitated 44 h later, and brain tissue was removed. VMN was punched out as previously described (29). MBD and half of the remaining brain were used as positive controls. Total RNA was synthesized for RT-PCR as described above using the above primers for SRC-2 and SRC-3 and quantified by spectrophotometer (Ultraspec 2000, Pharmacia Biotech) at OD260. Each sample was normalized to total RNA for that sample, and data are expressed as arbitrary units. The experiment was repeated twice.
Compounds and Rat ER Oligonucleotides
All injections were prepared immediately before administration unless otherwise stated. Whenever possible doses were based on published studies for effective concentrations for reproductive behavior or verified when appropriate. Steroids were dissolved in sesame oil. Lyophilized oligonucleotides and human recombinant EGF were dissolved in sterile water; aliquots were stored at -70 until the day of use. The oligonucleotide sequences and their specificity for rat ER (AS, CAT-GGT-CAT-GGT-CAG; RS, ATC-GTG-GAT-CGT-CAC) have been reported previously (13).
Statistics
For behavioral studies, statistical analysis was performed using one-way ANOVA. Two-way ANOVA with repeated measures was used to assess significant change in reproductive behavior when animals served as their own control. Duncans multiple range test was used for individual comparisons.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: AS, Antisense; E, estrogen; EGF, epidermal growth factor; ER, estrogen receptor; icv, intracerebroventricular; IHC, immunohistochemistry; ir, immunoreactive; m, mouse; MBH, medial basal hypothalamus; P, progesterone; PR, progesterone receptor; r, rat; RS, randomized in sequence; SRC, steroid receptor coactivator; TBS-T, Tris-buffered saline/0.1% Tween; VMN, ventromedial nucleus.
Received for publication December 20, 2001. Accepted for publication March 12, 2002.
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REFERENCES |
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