Male ICR mice (age: 6–8 weeks, weight: 25–33 g) were bought from Yangzhou University Medical Center (Jiangsu, China) and subjected to a standard LD cycle (12L:12D) with lights on at 07:00 h and off at 19:00 h. Mice were offered a commercial mouse diet (Medicine, Jiangsu, China) and water ad libitum throughout the experiment. The protocol (from design to implementation) was in line with the methods put forward by the National Research Council’s Guide for the Care and Use of Laboratory Animals. Approval of the current work was provided by the Animal Protection and Utilization Committee of Yangzhou University (SYXK[Su] 2017-0044).

For in vitro experiments, 0.1% collagenase II (Sigma, St. Louis, MO, USA) and 0.25% pancreatic enzyme (Sigma, St. Louis, MO, USA) were used for digestion of freshly dissected mouse pituitary PD [20]. The cells of anterior pituitary were harvested and resuspended with Dulbecco’s modified Eagle medium (DMEM/F-12, GIBCO-Invitrogen, Grand Island, NY, USA), and 10% fetal bovine serum and antibiotics (penicillin G 100 IU/mL, streptomycin 100 µg/mL) were added, followed by cultured in 24-well plates at a density of 10⁵ cells/well. The cells were incubated at 37°C for 12 h in a mixture of 95% air and 5% CO₂. Each experiment involved at least three to four independent replicates, and each trial contained three to five replicates per treatment condition.

The CK1α agonist pyrvinium (MCE, Shanghai, China) was used to determine whether CK1α mediates the effects of melatonin on TSH synthesis in PD cells.

To explore the role of melatonin in CK1α expression and TSH synthesis, five mice were subjected to subcutaneous injection in the neck with 100 µL of 0.9% NaCl and 6 µg melatonin (Macklin, Shanghai, China) daily 1 h before lights off for 3 weeks. A control group of five mice was simultaneously injected with NaCl only [4].

Following in vivo and in vitro experiments, blood and cell culture supernatants were collected. The samples were centrifuged at 4,500 rpm, for 5 min at 4°C, then divided into equal aliquots, and stored at −20°C until use. TSH levels (pg/mL) in the stored samples were determined using an ELISA kit according to the manufacturer’s instructions (Adanti, Wuhan, China). The absorbance of each well was measured at 450 nm using a microplate reader. The detection range of the ELISA kit was 2–90 pg/mL (the final dilution of the sample was five times). The intra- and inter-determination coefficients of variation of the parameters were less than 9% and 11%, respectively.

Reagents (methanol, 0.1% (v/v) formic acid, ultrapure water, and ethyl acetate) were of chromatographic grade. After 3 weeks of melatonin administration, blood samples were collected for quantification. Fresh serum samples (300 µL) were treated with 1 mL of ethyl acetate, shaken for 3 min, then centrifuged at 13,400 × g and 4°C for 10 min. The supernatant was collected, dried under nitrogen for 15 min, then resuspended in 0.15 mL of a solution containing 65 parts of 0.1% (v/v) formic acid in ultrapure water and 35 parts of methanol. The mixture was swirled for 1 min and centrifuged for 10 min at 12,000 rpm and 4°C. 1 mL syringe was used to collect the supernatant. The supernatant was collected using a 1 mL syringe. Samples were separated and detected with high-performance liquid chromatography tandem mass spectrometry.

The mobile phase consisted of 0.1% (v/v) formic acid in ultrapure water (buffer A) and methanol (buffer B). Mass spectrometry procedures followed an established method, with peak injection and extraction times at approximately 3.75 min [21]. The melatonin standard was diluted two-fold, dissolved in methanol, and diluted in the mobile phase (A:B = 65:35) at 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.156, 0.078, 0.04, 0.02, and 0.01 ng/mL.

Total RNA was extracted from the pituitary PD or cultured pituitary PD cells using the TRIzol reagent (Takara, Dalian, China) according to the manufacturer’s instructions. Complementary DNAs were synthesized using M-MLV reverse transcription reagents (Vazyme, Nanjing, China). Gene expression levels were measured using SYBR Green master mix (Vazyme, Nanjing, China) in the ABI-PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Endogenous Gapdh was used for standardization. Finally, according to the Ct value, the relative expression was calculated by the 2^−ΔΔCt method. Suppl. Table S1 shows all primer sequences.

To conduct immunofluorescence and immunohistochemistry, the pituitary PDs were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5 µm pieces. The whole pituitary was cut into 15 pieces, and every three slices were stained. Next, sections were then dewaxed using xylene for 10 min before being incubated with 100%, 95%, 85%, 75%, and 50% ethanol for 5 min each at 25°C. Antigen retrieval was performed in 0.01 mol/L Sodium Citrate Buffer (pH6.0). Then, the sections were treated with 10% normal donkey or goat serum in a phosphate buffer solution (PBS) for 1 h at 25°C to block non-specific binding sites, incubated at 4°C overnight with primary antibody and washed with PBS (for antibodies, see Suppl. Table S2). Sections were then incubated with fluorescent conjugated secondary antibodies for 1 h at 25°C, then incubated with 4’, 6’-diamino-2-phenylindole (DAPI, Beyotime, Shanghai, China) for 10 min, and incubated again at 25°C. Finally, sections were visualized under an Olympus IX71 fluorescence microscope (Olympus, Tokyo, Japan). The number of TSH-β⁺, CK1α⁺, MT1⁺, TSH-β⁺-MT1⁺ and CK1α⁺-MT1⁺ cells was counted in five slices of each pituitary.

Proteins were extracted from the pituitary of the mice or the cultured primary pituitary cells by the radioimmunoprecipitation (Cell Signaling, Danvers, MA, USA) containing 1 mmol/L phenylmethylsulfonyl fluoride (PMSF; Cell Signaling, Danvers, MA, USA). Protein concentration was determined in accordance with the manufacturer’s instructions following a bicinchoninic acid assay (Vigorous Biotechnology, Beijing, China). The protein lysates were electrophoresed on 13% SDS-PAGE and then transferred to the polyvinylidene fluoride membranes (Bio-Rad Laboratories, Richmond, CA, USA), which were blocked for 1 h with 5% (w/v) non-fat dry milk, then incubated with antibodies for 12 h at 4°C. Then the membranes were washed thrice in Tris-buffered saline with Tween-20 and incubated with horseradish peroxidase (HRP)-conjugated donkey anti-goat IgG, HRP-conjugated goat anti-mouse IgG, or HRP-conjugated goat anti-rabbit IgG for 1–2 h at 25°C. After washing, the protein bands were visualized using a highly sensitive enhanced chemiluminescence detection kit (E412-01; Vazyme, Nanjing) at 2°C–8°C. Gray values were analyzed in ImageJ 1.53a. All antibodies are shown in Suppl. Table S2.

Data are presented as means ± S.E.M. of at least three independent experiments using GraphPad Prism 9.0 software. Student’s t-test was used for single comparison, and one-way analysis of variance was used for multiple comparisons; p < 0.05 was considered statistically significant, and p < 0.01 was considered extremely significant.

The results of RT-qPCR indicated that MTNR1A mRNA expression (encoding the MT1 receptor) was higher than MTNR1B mRNA expression (encoding the MT2 receptor) in the pituitary gland (Fig. 1A). Their respective proteins exhibited similar differences according to WB results (Fig. 1B). Immunohistochemistry then detected MT1 receptor signals in the mouse PD, whereas MT2 receptors signals were found in the pars intermedia (PI) (Fig. 1C). These findings suggest that melatonin is important in mouse PD hormone synthesis.

Melatonin is the main circadian-rhythm-regulating hormone in animals. We investigated the effects of melatonin administration on melatonin levels and PD-TSH expression in vivo using liquid chromatography, RT-qPCR, and WB. Exogenous melatonin increased melatonin by 10-fold from control levels (Fig. 2A). Furthermore, Tshb mRNA expression and TSH level increased two-fold and 1.6-fold from control levels in melatonin-treated mice respectively, whereas Mtnr1A mRNA expression did not change significantly (Figs. 2B–2D). Melatonin treatment increased MT1 protein expression by 1.3-fold from control levels (Fig. 2E).

These in vitro results are similar to the results described in in vivo. We treated primary pituitary cells with melatonin at different concentrations (0, 0.1, 1 and 10 μM) and at different timepoints (0, 5, 10 and 20 min). Treatment with 10 μM melatonin for 5 min increased Tshb mRNA and TSH level (Figs. 2F–2H).

Notably, immunofluorescence staining showed that 45% MT1 and TSH-β signals colocalized. Additionally, 90% of the MT1 and CK1α signals colocalized in the PD (Figs. 3A and 3B). Next, we used RT-qPCR and WB to measure the effects of melatonin administration on CK1α expression in vivo. Although melatonin did not significantly affect Csnk1a1 mRNA expression (Fig. 3C), it decreased the ratio of phosphorylated to total CK1α protein (p-CK1α/CK1α) by 1.6-fold from control levels (Fig. 3D).

These in vitro results are similar to the results described in vivo. We treated primary pituitary cells with melatonin at different concentrations (0, 0.1, 1, and 10 μM) and timepoints (0, 5, 10, and 20 min). In response to treatment with 10 μM melatonin for 5 min, Csnk1a1 mRNA and protein levels significantly decreased (Figs. 3E and 3F). We then determined whether melatonin regulated PD-TSH by acting on CK1α. The primary pituitary cells were pre-treated with CK1α activator pyrvinium for 6 h, followed by 5 min of melatonin treatment. These results suggest that pyrvinium blunted the upregulation of PD-TSH synthesis induced by melatonin (Figs. 3G and 3H).

We previously found that CK1α is highly expressed in pituitary TSH cells [19], and TSH is known to be rhythmic [22]. To explore possible variation in the relationship between CK1α and TSH, we first measured Tshb mRNA and CK1α protein expression under 12:12 light-dark conditions (LD; zeitgeber time ZT7-lights on and ZT19-lights off). Tshb mRNA expression in the PD varied during the day, peaking at ZT7 and dropping at ZT22 (Fig. 4A). CK1α protein levels also fluctuated during the daytime, with higher levels at ZT22 than at ZT7 (Fig. 4B). These results show that Tshb mRNA and CK1α protein expressions levels peaked at opposite phases of the 24-h light:dark cycle.

The PKC/ERK/CREB signaling pathway regulates pituitary TSH synthesis, and previously, we showed that CK1α modulates this signaling cascade to regulate TSH synthesis [19]. CK1α is a component of the β-catenin degradation complex and plays a negative regulatory role in Wnt/β-catenin signaling [23,24]. Therefore, we used WB to investigate circadian-related phosphorylation of key proteins in the PKC/ERK/CREB and Wnt/β-catenin signaling pathways. Phosphorylated PKC, ERK, and CREB levels in the mouse pituitary were significantly higher during the day (7:00) than at night (22:00) (Fig. 4C). Phosphorylated GSK3β in the Wnt pathway did not exhibit significant circadian variation, but ratios of active β-catenin and phosphorylated β-catenin to total β-catenin downstream of CK1α were significantly higher at night than during the day (Fig. 4D). Therefore, the activation of the CK1α-PKC/ERK/CREB and CK1α/Wnt/β-catenin signaling pathways is associated with the circadian rhythm of murine pituitary TSH synthesis.

We then examined the effect of melatonin on the PKC/ERK/CREB and Wnt/β-catenin signaling pathways. Results from WB indicated that exogenous melatonin enhanced p-PKC, p-ERK1/2, and p-CREB protein expression in PD (Fig. 5A) but had no effect on the mediators of Wnt/β-catenin signaling (Fig. 5B).

CK1α regulates the production of TSH by modulating the PKC/ERK/CREB pathway in pituitary tissues [19]. We verified whether melatonin affected PD-TSH synthesis in a CK1α-dependent fashion by acting on the PKC/ERK/CREB signaling pathway. After pre-treating primary pituitary cells with pyrvinium for 6 h, followed by 10 min of melatonin treatment, we observed that pyrvinium blunted melatonin-induced upregulation of phosphorylated PKC and CREB levels (Fig. 5C).