Animals

Two to three month-old female mice of the ICR strain (CLEA Japan Inc., Osaka, Japan) were used. They were kept in a temperature-controlled animal room (20–22°C; lights on, 07:00–21:00), and given a commercial diet and tap water ad libitum. All animal care and experiments were performed in accordance with the Guidelines of Animal Experimentation, Faculty of Science, Okayama University, Japan.

Pituitary cell dissociation and culture

Pituitary glands were removed, and the posterior and intermediate lobes were discarded. The anterior pituitary cells were isolated according to the method of Oomizu and Takahashi (1996). The dissociated cells were suspended at a density of 5 × 10⁵ cells/ml in an 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium (DMEM/F12, Sigma, St. Louis, MO, USA) with 10% fetal bovine serum. These cells were seeded in 24-well culture plates (Becton Dickinson, Uncoln Park, NJ, USA) and incubated at 37°C in water-saturated atmosphere of 5% CO₂ and 95% air for 3 to 4 days, before the cell immunoblot assay, in order to allow the cell damage caused by the enzymatic cell dispersion to heal. The cell viability was usually about 90% when assessed by trypan blue exclusion.

Cell immunoblot assay

Cell immunoblot assay was performed according to the method of Kendall and Hymer (1987) with slight modification. Briefly, the anterior pituitary cells were harvested from culture dishes using 0.02% trypsin-1 mM EDTA in Ca²⁺-Mg²⁺-free-Hanks' solution containing 20 mM HEPES and bovine serum albumin (0.3% w/v, BSA) at 37°C for 5 min and were dispersed into single cells (the cell viability was about 90%). These cells were suspended at the cell density of 2 × 10⁵ cells/ml in DMEM/F12 with supplement (hydrocortisone, 100 mg/ml; triiodthyronine, 400 ng/l; transferrin, 10 mg/ml; bovine glucagon, 10 ng/ml; parathyroid hormone 200 ng/l and sodium selenite, 5 mg/ml) and secretagogues (TRH and α-MSH) when needed. The cell suspensions were immediately used for the cell immunoblot assay. TRH was obtained from Peptide Institute (Osaka, Japan), and α-MSH from Sigma.

The polyvinylidene difluoride transfer membranes (21 × 28 mm, Immobilon, Millipore, Bedford, MA, USA) were used for the assay. They were prewetted in methanol for 30 sec, rinsed in distilled water for 5 min and equilibrated with DMEM/F12 for 60 min before cell blotting. The membranes were placed on plastic film with their water-repellent side up. One hundred microliters of the cell suspension was dispersed on the membranes, and a cover slip (18 × 24 mm) was gently placed on the cell suspension. The cell suspension was spread into the cover slip's area. Cells on the membranes were never broken by this operation. The membranes were incubated at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. After the incubation, the membranes were rinsed gently in 0.01 M phosphate-buffered saline for 15 min, then treated with 1% skim milk at 4°C for overnight to block unoccupied sites.

The PRL absorbed on the transfer membranes was immunostained, using rabbit antibodies generated against mouse PRL (Shikibo, Kusatsu, Japan) by the avidin-biotin-peroxidase complex technique (Hsu et al., 1980). The specificity of antibodies to mouse PRL was ascertained (Takahashi et al., 1994). Immunoreactivity of PRL was finally visualized using 4-chloro-1-naphthol as chromogen. After the color development, the membranes were rinsed twice with distilled water for 30 min and dried in air. The specificity of cell immunoblot was examined by omitting the antibody and by replacing the antibody with normal rabbit serum. In both cases immunoreactive PRL was not detected.

Image analysis

The immunoreactivity detected on the membrane was analyzed with a light microscope equipped with a CCD video camera (SONY XC-009P, Tokyo, Japan) and VIDAS Image Analysis System (Zeiss/Kontron, Germany) as follows. (1) Using a CCD video camera a black-and-white digital image of the membrane was produced at a resolution of 512 × 512 pixels, 8 bits/pixel = 256 gray levels. (2) The background gray values were measured. (3) The immunostained PRL spots were detected, and displayed on a monitor display. Too small objects or the objects touching edge of the image frame were discarded. (4) The non-PRL spots, for example colored scratch or too irregular shaped objects were also discarded. (5) Average value of gray scale and number of pixels of each spot were measured. The amount of PRL secreted was determined as follows: PRL amount = {(average gray value of PRL spot) – (back ground gray value)} × the number of pixels of each spot. PRL amount measured was expressed as percentages by regarding the amount of controls as 100%. In each assay 300 blots (PRL cells) were measured, and three independent assays were performed.

In our preliminary study, serially diluted pituitary extracts were applied to the transfer membrane, and immunostained to visualize PRL absorbed. The amount of PRL contained in the pituitary extracts was analyzed in our image analysis system. The PRL amount estimated was closely correlated with concentrations of pituitary extracts. We concluded that the amount of PRL secreted was able to be estimated using our cell immunoblot assay system.

Statistical analysis

Statistical differences in the average among groups were analyzed with a one-way analysis of variance followed by the multiple range test of Duncan. Statistical differences in the frequency distribution of PRL levels among groups were analyzed with KolmogorovSmirnov's two-sample test.

PCR primers

Oligonucleotide primers for specific amplification of each MC receptor were designed based upon nucleotide sequences of mouse MC receptors reported previously (Mountjoy et al., 1992; Desarnaud et al., 1994; Labbe et al., 1994; Kubo et al., 1995). Since the sequence of mouse MC-4 receptor gene was not available in DNA data bases, primers for MC-4 receptors were determined based upon the report by Boston and Cone (1996). Amplification of MC-3 receptor cDNA and MC-5 receptor cDNA shared a common primer pair. The presence of a MspI site in the amplified DNA fragment of the MC-5 receptor enables to distinguish it from that of the MC-3 receptor. The primer sequences were as follows:

Reverse transcription and PCR

Genomic DNA from newborn mice and total RNA from anterior pituitaries of adult female mice were prepared following the method described (Sambrook et al., 1989). Approximately 2 μg of the RNA was subjected to reverse transcription using Superscript II Reverse Transcriptase (Gibco BRL, Gaithersburg, MD, USA) according to the manufacturer's instructions. One tenth aliquot of the reaction was used in each PCR using primer pairs specific for each of MC receptors. In the case of PCRs using genomic DNA as a template, 0.8 μg of DNA was used in each PCR. The PCRs were carried out using AmpliTaq Gold DNA polymerase (PERKIN ELMER, Forster City, CA, USA) and a thermal cycler (Gene Amp PCR System 9600, PERKIN ELMER). The conditions for the PCR were as follows: after activation of the DNA polymerase by incubating for 9 min at 95°C, 40 cycles of reactions including denaturation for 30 sec at 95°C and extension for 1 min at 60°C were performed, followed by additional extension for 10 min at 60°C. One tenth aliquot of each reaction was electrophoresed on 2.0% agarose gel, stained with ethidium bromide, and photographed under ultraviolet illumination. Each of the amplified DNA fragments were subcloned into pGEM3Zf(+) and subjected to sequencing. Dideoxynucleotide sequencing was performed using fluorescent primers and an automated DNA sequencer (Applied Biosystems 373A).

Effects of TRH and α-MSH on PRL release

The relative amount of PRL released during 5-hr incubation is illustrated in Fig. 1. The PRL amount increased as incubation was prolonged. The rate of PRL secretion decreased between 2 and 3 hr of incubation. Both TRH (10⁻⁷ M) and α-MSH (10⁻⁷ M) treatment stimulated PRL secretion compared with controls. In the following experiments, 1 hr-incubation was used for the measurement of PRL secretion.

Fig. 1

Effects of TRH (10⁻⁷ M) and α-MSH (10⁻⁷ M) treatment on PRL secretion from single cells of adult female mouse pituitaries during incubation. Each point and bars depict the mean and its standard error of the PRL amount released from about 300 PRL cells. TRH increased PRL release compared with corresponding controls (** p < 0.01). α-MSH increased PRL release compared with controls (# p < 0.05, ## p < 0.01).

TRH significantly increased the amount of PRL released in a dose-dependent manner (10⁻¹¹ – 10⁻⁷ M, Fig. 2). Thus, stimulatory effect of TRH on PRL release was confirmed in our system, indicating that the conditions of cell immunoblot assay in our study are suitable for analyzing PRL release.

Fig. 2

Effects of TRH on PRL secretion from single cells of adult female mouse pituitaries during 1-hr incubation. Each column shows mean of three independent assays with standard errors. * p < 0.05, ** p < 0.01.

As in the case of TRH, α-MSH significantly increased the amount of PRL released in a dose-dependent manner (10⁻¹¹ – 10⁻⁷ M, Fig. 3). Figure 4 shows the typical frequency distribution of the amount of PRL release from single PRL cells in response to α-MSH of various concentrations (10⁻¹¹ – 10⁻⁷ M). The frequency distribution of the amount of PRL release displayed a unimodal right side-trailing pattern at any doses of α-MSH. Although the mode of the frequency was not affected, the median value of the PRL amount at 10⁻⁷ M α-MSH was shifted rightward compared with controls.

Fig. 3

Effects of α-MSH on PRL secretion from single cells of adult female mouse pituitaries during 1-hr incubation. Each column shows mean of three independent assays with standard errors. * P < 0.05, ** P < 0.01.

Fig. 4

Typical frequency distribution patterns of PRL secretion from single cells of adult female mouse pituitaries during 1-hr incubation. About 300 blots (PRL cells) were measured in all groups. The arrow indicates the median value of PRL release. The distribution pattern of PRL secretion in α-MSH treatment (10⁻⁷ M) statistically differed from that in control (P < 0.01).

Detection of MC receptors by RT-PCR

Evaluation of the specificity of primers used for amplification of each MC receptor was carried out by a series of PCRs using genomic DNA as a template and sequencing. As shown in Fig. 5A, each primer pairs amplified DNA species with the expected length. Digestion of PCR-products with MspI showed that the primer pair used for both the MC-3 receptor and the MC-5 receptor can amplify both genes with the same quantity (Fig. 5B). Sequence analysis of each PCR-amplified DNA fragment revealed that they were identical to the reported MC receptors (data not shown). The PCR using MC-4 receptor primers generated two species of DNA fragments. The nucleotide and predicted amino acid sequence of the larger DNA fragment (554 bp) shared 90.0 and 95.1%, respectively, identity with those of the human MC-4 receptor. On the other hand, the smaller DNA fragment (263 bp) was observed even in the absence of the template (data not shown), and had no homology with the human MC-4 receptor, suggesting that the former is the mouse MC-4 receptor gene and the latter is an artifact of PCR. The sequence of the mouse MC-4 receptor is available from DDBJ, EMBL, and GenBank Data Libraries under the accession number AB009664.

Fig. 5

Evaluation of the specificity of oligonucleotide primers. Primers specific for each MC receptor were designed and tested against genomic DNA. (A) Specific amplifications of MC receptor genes. Products of PCRs using primer pairs specific for the MC-1 receptor (lane 1), the MC-2 receptor (lane 2), the MC-3/5 receptors (lane 3) and the MC-4 receptor (lane 4) were electrophoresed. (B) Restriction patterns of the DNA fragments obtained by PCR using the MC-3/5 receptors-specific primer pair. The amplified DNA fragments were electrophoresed intact (lane 1) or after digested with MspI (lane 2). In each case, a 100 bp ladder was used as a molecular marker.

Using these specific primers for the mouse MC receptors, total RNA prepared from anterior pituitaries of adult female mice were analyzed by RT-PCR. As shown in Fig. 6A, the amplification of DNA fragments were only observed in PCR using the MC-3 receptor /MC-5 receptor primers. Inability of the PCR-product to be digested with MspI (Fig. 6B) suggested that only MC-3 receptor cDNA was amplified by the PCR. In order to confirm it, we performed sequencing of the DNA fragment and found that it was indeed a cDNA for the mouse MC-3 receptor (data not shown). These results indicate that the MC-3 receptor is the sole MC receptor expressed in anterior pituitaries of adult female mice.

Fig. 6

Identification of the type of MC receptors expressed in anterior pituitaries of adult female mice. (A) RT-PCR analysis of MC receptor expression in anterior pituitaries of adult female mice. Reaction mixtures of RT-PCRs using primer pairs specific for the MC-1 receptor (lane 1), the MC-2 receptor (lane 2), the MC-3/5 receptor (lane 3) and the MC-4 receptor (lane 4) were electrophoresed. (B) Restriction patterns of the DNA fragments obtained by RT-PCR using MC-3/5 receptor-specific primer pair. The amplified DNA fragments were electrophoresed intact (lane 1) or after digested with MspI (lane 2). In each case, a 100 bp ladder was used as a molecular marker. MspI failed to digest the RT-PCR product, indicating that cDNA for the mouse MC-3 receptors was amplified.