Animals

BALB/c mice were purchased from CLEA Japan, Inc. (Tokyo, Japan) and housed in plastic cages with wood shavings under controlled temperature (25 ± 0.5°C) and lighting (12 hr from 6:00 to 18:00). They were given a commercial diet (CE-7; CLEA Japan) and tap water ad libitum. All procedures used on the mice were described in detail in a protocol that was approved by the Animal Care and Use Committee of the Graduate School of Science, University of Tokyo, and all experiments conformed to the regulations described in the NIH Guide to the Care and Use of Laboratory Animals.

The mice were killed at 2 months of age without further treatment. In addition, hyperprolactinemic mice were prepared as described (Matsuda et al., 1995). Two-month-old female mice were transplanted with a single anterior pituitary gland obtained from male litter-mates under the left kidney capsule (PG mice), or were sham-operated as the control group. These mice were sacrificed at 15 days after the operation, when the circulating level of prolactin increased to more than 10-fold the control value (Matsuda et al., 1994).

Preparation of cDNA samples

Tissues were dissected from decapitated mice and stored in liquid nitrogen until use. Total RNA was isolated from the frozen tissues using acid guanidinium-phenol-chloroform method (Chomczynski and Sacchi, 1987). The concentration of total RNA was estimated by the absorbance at 260 nm (1 OD unit was estimated at 40 μg/ml) using a spectrophotometer (Ubest-30; Japan Spectroscopic Co., Ltd., Tokyo). After denaturation at 90°C for 5 min, the RNA samples were ice-chilled, then template cDNAs for PCR were generated from the samples by RT according to standard procedures. RT of 2 μg total RNA was performed in a 20-μI reaction mixture containing 1× RT buffer (72 mM KCl, 3 mM MgCI2, 50 mM Tris-Cl, pH 8.3), 10MM dithiothreitol (DTT), 1 mM each of the four deoxyribonucleotide triphosphates (dNTP; Pharmacia LKB, Sweden), 20 U of ribonuclease inhibitor (RNasin; Promega, Madison, Wl), 200 U of Superscript^™ reverse transcriptase (GIBCO BRL, Gaithersburg, MA) and primers. The primers were 5 (μM random hexamers (Takara Inc., Tokyo) or 100 nM oligo deoxythymidine (oligo d(T)_12-18; Pharmacia). The reaction mixture was incubated at 23°C for 10 min (annealing), at 42°C for 1 hr (elongation), at 95°C for 10 min (deactivation), then ice-chilled and divided into 2.5 μl of aliquots.

PCR primers and conditions

Eight oligonucleotides were synthesized (Sawaddy Technology, Inc., Tokyo) as PCR primers, designed to amplify the extracellular or cytoplasmic regions of mPRL-R cDNAs and to generate mPRL-R cDNA bearing a point mutation in the extracellular regions. The priming regions of cDNA synthesis using a truncated model of the two forms of mPRL-R mRNA, PRL-R_S3 and PRL-R_L1 (Clarke and Linzer, 1993) are shown in Fig. 1.

Fig. 1.

A truncated model of two forms of PRL-R mRNAs (PRL-R_L1 and PRL-R_S3), regions of eight PCR primers, and DNA fragments amplified by PCR. The two forms have a common 5′ flanking region and sequences corresponding to the signal peptide (S), extracellular domain (EC), transmembrane domain (TM) and part of the intracellular domain (IC). They differ in the 3′ region corresponding to IC (indicated with striped box) and in the 3′ flanking region. EC has a BamHl site at which double-strand cDNA is digested by the RE. IC of the long or short forms contains Mspl site or BamHl site, respectively. Arrowheads indicate regions of PCR primers. P1, mPRLR-1; P2, mPRLR-2; P3K, mPRLR-3K; P4, mPRLR-4; L1, mLTHR-1; LL1, mLTHR-L1 A; LS3, mLTHR-S3A; L3K, mLTHR-3K. Bold lines below indicate DNA fragments amplified by PCR, and the names and sizes are indicated above or on the left of them. Vertical thin lines indicate RE sites, and the sizes of cleaved fragments are indicated in parentheses.

The sequences of primers were as follows: mPRLR-1; 5′-CTG AAGGGAGCCTCTGATCTATTGC-3′, mPRLR-2; 5′-GGATTTGATA CTCATCTGCTAGAG-3′, mPRLR-3K; 5′-CCTCGGTACCACTTTAT GTG-31, mPRLR-4; 5′-ATGCCATCTGCACTTGCTTACATG-3′, mLTHR-1; 5′-GATTTCTCCTGGCCCCATCTACTCC-3′, mLTHR-L1 A; 5′-CATAGCTTCCATGACCAGAGTCAC-3′, mLTHR-S3A; 5′-GCAT CCTTGAGACTAGATTATTGG-31, mLTHR-3K; 5′-ACATAAAGT GGTACCGAGGT-3′ (Kpnl sites are underlined).

The cDNA sample was amplified by PCR in a 40-μI reaction mixture containing 1× PCR buffer (50 mM KCl, 1.5 mM MgCI₂, 10 mM Tris-Cl, pH 8.0), 200 μM each of dNTP, and primers as indicated below in each reaction. Sometimes, 148 kBq of deoxycytidine 5′-[α-³²P]triphosphate ([α-³²P]dCTP) (Amersham, England) was added to radioisotopicaily label the PCR products. The reaction mixture was overlaid with mineral oil (Sigma, St Louis, MO), and hot start PCR amplification proceeded using a thermal cycler (PTC-100; MJ Research, Inc., USA). After denaturation at 90°C for 5 min, 1 U of Taq DNA Polymerase (Takara) was added to the reaction mixture, then 40 cycles of PCR amplification were performed. Each cycle consisted of denaturation at 94°C for 1 min, annealing at 62°C for 1 min, and elongation at 72°C for 1 min.

Preparation of competitor and standard DNA for cPCR and standard DNA for one-sided cPCR

Fragments of mPRL-R cDNA were amplified from mouse liver cDNA by PCR using three sets of primers; 500 μM each of mPRLR-1 and mLTHR-L1A, mPRLR-2 and mLTHR-L1A, and mPRLR-2 and mLTHR-S3A. The PCR reaction mixture was resolved by electrophoresis on 1 or 2% agarose gel in1×TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.0). The gels were stained with ethidium bromide (EtBr), and the PCR products were visualized with a UV transilluminator. Gel strips containing a single PCR product of the appropriate size (1108, 232 or 228 bp, respectively) with a low background were excised. DNA was extracted from the gel using glass powder (Easytrap^™; Takara, Inc.) and subcloned into the pT7Blue(R)-T vector (Novagen, Madison, Wl) (mPR1-L1A(+)pT7B, mPR2-L1A(+)pT7B or mPR2-S3A(+)pT7B, respectively). XL1-Blue (Stratagene, La Jolla, CA) was the host bacterium. The insert DNA was isolated from the plasmid by digestion with restriction enzymes (REs), EcoRI and Pstl, subcloned into pUC118/119 vectors, then sequenced as described (Akazome et al., 1994). We confirmed that they were derived from the target fragment of mPRL-R cDNAs. All REs used here were obtained from Takara.

PCR was also used to generate a mutant of mPR1-1LA(+)pT7B bearing a Kpnl site (GGTACC) instead of a BamHl site (GGATCC) in the region corresponding to the extracellular domain of mPRL-R. MPR1-1LA(+)pT7B was linearized with Hindlll, and PCR amplified using mPRLR-4 and mLTHR-3K or using mPRLR-3K and mLTHRL1A as primers. Each PCR product was subcloned into pT7Blue(R) vector (mPR4-3K(+)pT7B and mPR3K-L1A(+)pT7B). Both plasmids were digested with Kpnl, and the mPR4-3K(+)pT7B digest was dephosphorylated using calf intestine alkaline phosphatase (Takara) and ligated with the smaller fragment of mPR3K-L1 A(+)pT7B digest to generate the mutant mPR1 -1 LA(+)pT7B bearing a Kpnl site (mPR1 -1LA(K)(+)pT7B).

Both plasmids, mPR1-1LA(+)pT7B and mPR1-1LA(K)(+)pT7B, were digested with Pstl and EcoRI, extracted with phenol-chloroform and precipitated with ethanol. They were then quantified by absorption at 260 nm (1 OD unit was estimated at 50 μg/ml), and used as the standard and the competitor DNA for cPCR, respectively.

Competitive PCR for the determination of rough amount of PRL-R cDNAs

PRL-R cDNA content was estimated by cPCR of cDNA samples (or standard DNA) with 10³ or 10⁷ molecules of competitor DNA using 500μM each of mPRLR-4 and mLTHR-1 as primers. The PCR product derived from the competitor bearing Kpnl site was distinguished from that from the tissue-derived native cDNA or the standard DNA bearing BamHI site by REs digestion. After digestion with either or both enzymes, the PCR products were resolved by electrophoresis on a 4% Polyacrylamide gel in 1× TBE buffer (50 mM Tris, 50 mM boric acid, 1 mM EDTA, pH 8.0) and stained with EtBr.

One-sided cPCR for the determination of S/L ratio

PCR amplification of cDNAs proceeded in the presence of [α-32P]dCTP using 100 μl of mPRLR-2 and 500 μM each of mLTHRL1A and mLTHR-S3A as primers. Smaller amount of the common primer (mPRLR-2) than the identical primers (mLTHR-L1A and mLTHR-S3A) reduced potential alteration of the S/L ratio caused by exhaustion of the identical primers. The PCR product derived from the long form was distinguishable from that from the short form by having a restriction site for BamHl or Mspl. The PCR mixture was digested with the enzymes, resolved by electrophoresis on 10% Polyacrylamide gel in 1× TBE buffer, and stained with EtBr. The band corresponding to the larger fragment of digested PCR product was excised under UV light, transferred into a glass vial, then Cerenkov radiation was measured using a scintillation counter (LS60001C; Beckman Instruments, Inc., Fullerton, CA). The PCR product without RE digestion was also resolved by electrophoresis, and a gel strip equivalent to the larger fragment of digested PCR product at the same position and size served as the background. The value of radiation intensity of gel corresponding to the larger fragment of Mspl or BamHl digest was divided by the number of deoxycytidine residues in the alignment (83 or 77, respectively). The quotient was proportional to the number of long or short form mPRL-R cDNA molecules. Sometimes, the gels were exposed to a Kodak X-OMAT film (Eastman Kodak, USA) after electrophoresis.

Statistical analysis

All results are expressed as the mean ± S.E. Statistical significance of differences between means were assessed with Student's t-test. The number of samples is expressed as ‘n’ in parenthesis.

One-sided cPCR with standard DNA samples

To understand the relationship between the S/L ratio of one-sided cPCR products and that of the cDNA before amplification, one-sided cPCR was performed with samples containing fixed amounts of standard plasmid DNAs, mPR2-L1A(+)pT7B and mPR2-S3A(+)pT7B linearized by EcoRI digestion. After preparing standard samples containing 10³ or 10⁷ molecules in each tube and an S/L ratio of 0.1, 0.25, 0.5, 1, 2, or 4, one-sided cPCR amplification of mPRL-R cDNA was performed and the S/L ratio of the one-sided cPCR products was measured (Fig. 2). The results showed that S/L ratio was not altered by one-sided cPCR amplification. Hence, one-sided cPCR was considered to be reliable for measuring the S/L ratio, at least when it was between 0.1 and 4 and the amount of mPRL-R cDNA molecules was between 10³ and 10⁷.

Fig. 2.

One-sided cPCR products and its S/L ratio obtained from amplification of standard DNA for long and short form mPRL-R (mPR1-L1 A/pT7B and mPR1-S3A/pT7B, respectively) combined in various initial proportions. a: After digestion with BamHl (B) or Mspl (M), amplification products were resolved on a 10% Polyacrylamide gel and autoradiographed. The initial S/L ratio and RE are indicated on the top of the lanes. The amplification generated 232 and 228 bp products that corresponded to the long and short form mPRL-R, respectively. The product from the long form was digested by Mspl into 170 and 62 bp fragments, and that from the short form was digested by BamHl into 175 and 53 bp fragments. b: Relationship between initial S/L ratio and S/L ratio determined by one-sided cPCR after PCR amplification. The initial amount of standard DNA molecules was 10³ (○) or 10⁷ (•). Vertical bars indicate S.E. (n =3-4).

Competitive PCR for determination of rough amount of mouse PRL-R cDNA

To reconfirm that the number of PRL-R cDNA molecules was between 10³ and 10⁷ in the cDNA sample, we developed cPCR with which to estimate the amount of mPRL-R cDNA. Competitive PCR amplification proceeded with samples containing 10³ or 10⁷ molecules of standard DNA (mPR1-L1 A(+)pT7B) and competitor (mPR1-L1 A(K)(+)pT7B) (10^2-4 or 10^6-8 molecules, respectively) (Fig. 3A). The amount of PCR product cleaved by BamHl was more than by Kpnl when there was more standard than competitor and vice versa. We then performed cPCR of each cDNA sample using 10³ or 10⁷ molecules of standard (cf. Fig. 3B). All cDNA samples used here contained more than 10³ and less than 10⁷ molecules of mPRL-R cDNA.

Fig. 3.

Estimation of mPRL-R cDNAs in cDNA by cPCR. Left panel: cPCR of standard DNA (10^2-8 molecules/tube) with the competitor (10³ or 10⁷ molecules/tube). Right panel: cPCR of a hepatic cDNA sample with competitor cDNA (10³ or 10⁷ molecules). PCR products were digested with BamHl (B) or Kpnl (K) and resolved on a 4% Polyacrylamide gel. The amplification generated a 653 bp product, some of which was cut into two fragments (329 and 324 bp) by the REs. The amount of PCR products digested by BamHl was more than that by Kpnl when the amount of standard DNA (or native mPRL-R cDNA in cDNA sample) was more than that of the competitor, and vice versa. The amount of native mPRL-R cDNA in the cDNA sample is more than 10³ and less than 10⁷ molecules.

S/L ratio in several tissues

The S/L ratio of cDNA in the liver, mammary gland, ovary, uterus, testis, small intestine, skeletal muscle and blood was determined by one-sided cPCR (Fig. 4). Oligo dT primer was used for the RT reaction. The S/L ratio was above 1 in the liver, but less than 1 in the mammary gland, ovary, uterus, testis, small intestine and skeletal muscle. No PCR product was observed in the blood. However, sufficient amount of one-sided cPCR product was obtained from cDNA of the buffy coat which was separated by 1% dexistran from heparinized blood. We found that the long form was also dominant in the blood (data not shown). Thus, long form PRL-R mRNA is dominant in almost all mouse tissues except the liver.

Fig. 4.

One-sided cPCR products and the S/L ratio obtained from amplification of cDNA from tissues. Amplification products of cDNA from the small intestine, uterus, ovary, mammary gland, liver, skeletal muscle and blood of female mice and testis of males were digested by Mspl (M) or BamHl (B), resolved on 10% Polyacrylamide gels and autoradiographed. Means ± S.E. of their S/L ratios are indicated on the bottom of the lane (n = 3-4). ND, not detected. The long form PRL-R was dominant only in the liver.

Effect of PG on S/L ratio in liver

Hepatic cDNA samples were prepared in PG and control mice by the RT reaction using oligo dT primer or random primer. The S/L ratios then determined by one-sided cPCR (Fig. 5), were above 1 even when the random primer was used, suggesting that the short form was dominant in the liver. The S/L ratio was consistently higher in PG mice than in controls. Therefore, PG increased the amount of the short, rather than the long form of PRL-R mRNA in the mouse liver.

Fig. 5.

Effect of PG on the S/L ratio in female mouse liver. Hepatic cDNA samples were prepared from female PG (black column) and control (clear column) mice by RT using oligo dT (a) or random hexamer (b) as the primer, and the S/L ratios were measured by one-sided cPCR. Vertical bars indicate S.E. (n = 4). Statistical significance; *p<0.05.