A total of 60 rats were used in this study (the number of animals in each experiment can be found in Supplementary Table 1). All animal procedures in this study followed the guidelines of the European Communities Council (86/609/ECC) and were approved by the Regional Ethical Committee on Animal Research, Malmö/Lund, Sweden (M17-15) and/or the Danish Animal Experimentation Inspectorate. The animals were housed in Euro standard cages (Type VI with 123-Lid) in groups of 3 animals. Rats were given food and water ad libitum, lived under constant temperature (+ 22 °C) and humidity (55%) and were sustained at a 12/12-h light–dark cycle.

Totally 26 Wistar rats weighing approximately 250–300 g were divided in two groups (15 males and 9 females) and were used for immunohistochemistry and RT-qPCR in this study All animals were anaesthetized using CO2 and decapitated. TGs were carefully dissected and were either fixed in 4% paraformaldehyde (PF) in phosphate buffer saline (PBS, Sigma Aldrich, pH 7.2) for 3–4 h at room temperature or fresh frozen in liquid nitrogen. For immunohistochemistry the TGs were cryo-protected with Sörensen’s phosphate buffer (pH 7.2), gradient containing 10% and 25% sucrose overnight. Then, they were embedded in an egg albumin-based protein medium and sectioned at a thickness of 10 µm using a cryostat (Microm Cryo Star HM 560). Finally, the sections were collected on microscope slides (Superfrost™, Merck Chemicals and Life Science, Sweden) and stored at -20 ºC until use.

Sections from both male and female rats were washed and permeabilized in PBS containing 0.3% Triton X-100 (PBST) for 15 min. Thereafter, the tissues were blocked for non-specific binding of antibodies for 1 h at room temperature in blocking solution containing PBST, 1% bovine serum albumin (BSA), and 5% normal serum. The sections were incubated over night at 4 °C in moisturized chambers with primary antibodies (for details, see Table 1). The next day, the sections were washed in PBST for 3 × 15 min and incubated with secondary antibodies (for details, see Table 2) for 1 h at room temperature and kept in the dark to minimize loss of fluorescence. All antibodies were diluted in PBST containing 1% BSA. The sections were subsequently washed with PBST for 3 × 15 min and mounted with anti-fading Vectashield mounting medium containing 4', 6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame CA, USA). The described procedure was performed in triplicate for each animal to ensure reproducibility. Further, negative controls were included by omitting the primary antibody to evaluate auto-fluorescence and non-specific secondary antibody binding. Immunoreactivity was visualized using an epifluorescence microscope (Nikon 80i; Tokyo, Japan) at the appropriate wavelengths and photographed with an attached Nikon DS-2Mv camera. Images were processed using Adobe Photoshop CS3 (v0.0 Adobe Systems, Mountain View, CA).

Five different antibodies were selected to detect expression of PR, and two different antibodies were chosen to detect expression of progesterone in TG (Table 1). One antibody from ThermoFisher with catalog number MA5-12,658 was chosen to represent the visualization of the results of PR as well as antibody from Novus Biologicals (Cat.No. NB100-65167) was chosen to represent the visualization the results of progesterone in the figures.

Double immunohistochemistry was performed using antibodies against progesterone and PR in combination with CGRP and/or its receptor components calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1), or astrocyte and satellite glial cell marker glutamine synthetase (GI Syn). The antibodies were applied and mixed as a cocktail. All procedures were the same as described above.

Cell counting was performed to semi-quantify the expression of progesterone in TG. Three slides with three sections on each were used for measurements. Counting of cells which had visible nuclei was performed in the thickest part of pooled ophthalmic-maxillary and mandibular areas. Due to the risk of artefactual fluorescence, counting of neurons close to the TG surface was not performed. Images were taken of the screen (0.75 mm2) at 10 × magnification. A NIS-elements BR image analysis program (Nikon) was used to calculate the number of cells and to measure the fluorescence intensity in each area. All cells in this area, including negative and immunoreactive cells, were counted. The mean percentage of positive neurons in 3 slides/rat, from all rats (n = 6) was used for analysis. The intensity measurements were used to verify that immunoreactive cells were correctly distinguished from negative cells.

We performed two different experiments with real-time quantitative PCR (RT-qPCR). First, rat hypothalamus and TGs from eight male rats were carefully dissected, and immediately frozen in liquid nitrogen for RNA extraction. All RNA extraction was performed using RNeasy® Plus Mini kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s protocol. Total RNA concentration was determined using a GeneQuant Pro spectrophotometer (Amersham Pharmacia Biotech, Uppsala, Sweden). A ratio of sample absorbance at 260 /280 nm in the range of 1.8 to 2 was considered acceptable. First-strand cDNA was prepared from 1 µg of total RNA (from hypothalamus and TG) in a 20 µL reverse transcript reaction using Superscript® III First-Strand Synthesis Super Mix (Invitrogen, Carlsbad, CA, USA). A reverse transcription negative control for each sample to detect the genomic DNA was performed simultaneously and underwent the same procedures but without Superscript III Reverse Transcriptase (RT enzyme). The cDNA obtained was diluted to a total volume of 80 µL and stored at -20 °C. The primer sequences were specific for the genes of interest and were designed using Primer Express 3.0 software (PE Applied Biosystems, Foster city, CA, USA) and synthesized by TAG Copenhagen A/S (Copenhagen, Denmark). The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene and the gene expressions were normalized versus that. Primers had the following sequences:

PR (A + B) (forward; 5`- CTGCTGGATGAGCCTGATGGTG-3`, revers; 5`- CACCATCCCTGCCAGGATCTTG-3`); PR-B (forward; 5`-CAGACCAACCTGCAACCAGAA -3`, revers; 5`-AGTCCTCACCAAAACCCTGGG-3`); GAPDH (forward; 5`-CTGCACCACCAACTGCTTAGG -3`, revers; 5´-TCAGCTCTGGGATGACCTTGC- 3`).

The RT-qPCR was performed in 20 µL reaction consisting of 2 µL diluted cDNA, 0.6 µM of each primer, 10 µL Fast SYBR™ Green Master Mix (Applied Biosystems, CA, USA), and 7 µL RNase free water in a Step One Plus Real Time PCR System (Applied Biosystems, CA, USA) with the following thermal profile: Holding stage at + 50 °C for 2 min, + 95 °C for 10 min, followed by 40 PCR cycles at + 95 °C for 15 s and + 60 °C for 1 min. Each sample was examined in duplicate, and a blank control (without template) was used in all experiments. After amplification a melting curve analysis was performed to confirm specificity of primers annealing and to verify that each primer pair generated only one PCR product of expected size.

The second experiment was designed to analyze the expression level of PR in three male and three female rats. Total RNA was extracted from the TG using spin columns (NucleoSpin miRNA, Mini kit for total RNA, MACHERY-NAGEL) in combination with QIAzol (Qiagen, Germany) and chloroform (Sigma Aldrich, Denmark). The samples were homogenised using QIAzol lysis buffer (Qiagen, Germany) and 1.4 mm ceramic beads (Lysing Matrix D, MP Biomedicals, USA) for 40 s at max speed using a FastPrep-24TM 5G instrument (MP Biomedicals, USA). The RNA concentration was measured using a Nanodrop 2000c (ThermoFisher, USA) at 260 nm. 1 µg RNA was reverse transcribed using the iScript cDNA Synthesis Kit (Biorad, USA) according to the manufacturer´s protocol. RT-qPCR was performed using 20 × pre-designed TaqMan rat specific gene expression assay (IDT, USA), Prime Time Gene expression Master mix, and analysed using the Quant-Studio 6 Pro Real-Time PCR system (Applied Biosystems, USA). The thermal cycling condition included an initial denaturation step at + 50 °C for 2 min and + 95 °C for 10 min followed by 40 PCR cycles at + 95 °C C for 15 s and + 60 °C for 1 min. Pre-designed TaqMan gene expression assays used in this study detects all transcript variants and were purchased from IDT: PGR: Rn.PT.58.10589420 and ACTB: Rn.PT.39a.22214838.g.

Additional five male and five female rats were subjected to anaesthesia using CO2 before decapitation. TGs were carefully dissected and immersed in a 10 mL solution of synthetic interstitial fluid (SIF), comprising 108 mM NaCl, 3.5 mM KCl, 3.5 mM MgSO4, 26 mM NaHCO3, NaH2PO4, 1.5 mM CaCl2, 9.6 mM NaGluconate, 5.6 mM glucose, and 7.4 mM sucrose, maintained at a temperature of + 37 °C for a period of 30 min. Subsequently, these TGs were transferred into Eppendorf tubes positioned on a + 37 °C heating block and subjected to a rinsing process of four cycles, each lasting for 10 min with 300 µL of SIF, similar as previously published [24, 27].

To probe the release from the dura mater, the hemispheres of the brain were carefully excised from the cranium while keeping the cranial dura attached to the skull. The skull halves were then placed into a container filled with 250 mL of SIF and subjected to a dual rinse cycle, each lasting for 15 min. Both skull halves were maintained in a humid chamber placed above a water bath to ensure a constant temperature of + 37 °C. The skulls were then washed four times with 300 µL SIF, with each wash lasting 10 min.

After a 15-min incubation period with 300 µL SIF, samples of 200 µL for measuring basal CGRP release were obtained from all tissues, combined with 50 µL enzyme immunoassay buffer, and preserved at -80 °C for future analysis within two weeks from the time of the experiment. Prior studies have validated the absence of significant variations between basal CGRP release from left and right tissue sides, enabling the use of one side for progesterone testing, while the other served as a vehicle control. This paired design was leveraged to minimize experimental and biological variations within this assay [28, 29].

The CGRP release from the TG samples and the cranium halves were initially assessed under basal conditions, followed by an evaluation under the influence of progesterone (Cat. No. 2835, Biotechne, Ireland) dissolved in DMSO (Sigma-Aldrich, Germany) after a 15-min incubation to test for potential progesterone-induced release. Subsequently, in the presence of the same concentrations of progesterone or vehicle (DMSO), 100 nM capsaicin (211,275, Sigma-Aldrich, Germany) was utilized to stimulate the release of CGRP. Prior experiments have determined that 10-min incubation duration is sufficient for significant and reproducible CGRP release above baseline levels [29]. The workflow can be found in Supplementary Fig. 1. CGRP release was quantified using commercial Enzyme Immunoassay (EIA) kits (SPIbio, Paris, France). The CGRP concentration was calculated based on the standard curve, and the data was normalized against its contralateral control. The CGRP EIA reagent contains antibodies directed against human-CGRP but demonstrating equal reactivity with rat and mouse CGRP. The protocol adhered to the manufacturer's guidelines. The optical density was measured at 410 nm using a microplate photometer (Tecan, Infinite M200, software SW Magellan v.6.3, Mannedorf, Switzerland).

Rats (n = 12 of either sex) were sedated with a mixture of O2/CO2 (30/70%) and euthanized by decapitation. Following euthanizing, the basilar artery was immediately extracted and submerged in a chilled, oxygenated physiological solution of Na-Krebs buffer (consisting of: NaCl 119 mM, NaHCO3 15 mM, KCl 4.6 mM, MgCl2 1.2 mM, NaH2PO4 1.2 mM, CaCl2 1.5 mM, and glucose 5.5 mM). Ring segments of approximately 2 mm in length were prepared for myography studies [30, 31] and mounted on 40 µm wires. These arterial segments were allowed a 30-min equilibration period before being expanded to their optimal lumen diameter L1 = 0.9 × L100, where L100 refers to the diameter of the vessel under a passive transmural pressure of 100 mmHg (13.3 kPa). Initial evaluation of the contractile capacity of each arterial segment was conducted by exposing them to 60 mM K+. Following this, a concentration–response curve for contractile (from baseline) or dilation (from a preconstruction with 100 nM U46619 (Cayman #16,450, BioNordika, Denmark) was developed via the cumulative addition of progesterone. U46619 is a potent thromboxane A2 analog that primarily functions as a vasoconstrictor, inducing smooth muscle contraction in blood vessels. Furthermore, the dilation response to capsaicin in the presence of 10 µM progesterone/vehicle (DMSO) was tested.