P0130

HEC-1A cells and JAR cells (American Type Culture Collection, Manassas, VA, USA) were grown in McCoy’s 5 A and RPMI 1640 medium. RL95-2 cells were grown in DMEM/F12 (1:1) with 0.005 mg/mL insulin. The three cells lines were all supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. When the cells reached 80% confluence, they were subsequently starved of serum for 3 h before treatment with P4 (Sigma, P0130), E2 (Sigma, E2758) or E2 and P4 (concentrations can be seen in figure legends) under serum-free conditions. Cells were harvested for RNA after treatment 48 h and for protein after treatment 72 h.

Females were subjected to weekly lordosis tests in a Plexiglas aquarium (37 cm long × 17 cm high × 21 cm wide). A sexually experienced male was placed alone in the aquarium and allowed to adapt for 15 min. Subsequently, 3 h after receiving a subcutaneous progesterone injection (500 µg, P0130, Sigma) to induce behavioral estrus, the lordosis responses of the female to the mounts of the stimulus male were recorded. The test lasted until the female received 10 mounts or 10 min had elapsed. For the first experiment (mating-induced Fos activation), ovary intact females were paired with males during 30 min. A lordosis quotient (LQ) was calculated by dividing the number of lordosis responses displayed by the female subjects by the number of mounts received (x100). Before each experimental condition (drug injection, cell ablation, and optogenetic stimulation), all females were subjected to at least three lordosis tests (with progesterone) in order to acquire sufficient sexual experience and thus a significant LQ. Tests were performed during the dark phase of the light cycle (5 h after lights out; for details, see ref. ^{67 (link)}). For all details on the different hormone treatments (estradiol vs. estradiol+progesterone), see Supplementary Table 1.

A 93 day waiting period from the first VCD injection was employed to ensure substantial ovarian follicular depletion (Lohff et al., 2005 (link); Acosta et al., 2009 (link); Koebele et al., 2020a (link)) prior to initiating daily hormone administration, modeling the early post-menopausal time point. Rats were then randomly assigned to one of the following treatment conditions: Vehicle (sesame oil; Sigma Aldrich S3547; n = 10), 17β-estradiol (E2; 3 μg/day; Sigma Aldrich, E8875; n = 10), Progesterone (PROG; 0.7 mg/day; Sigma Aldrich, P0130; n = 9), Levonorgestrel (LEVO; 0.6 μg/day; Sigma Aldrich, N2260; n = 9), E2 + PROG (3 μg E2 + 0.7 mg PROG/day; n = 10), or E2 + LEVO (3 μg E2 + 0.6 μg LEVO/day; n = 10) (summarized in Figure 1). All hormone treatments were dissolved in sesame oil, delivered via a 0.10 mL daily subcutaneous injection for 21 days prior to beginning behavioral testing. Hormone or Vehicle injections continued for the duration of the experiment until euthanasia.

Unless otherwise stated, females were ovariectomized in adulthood (>8 weeks of age) under general anesthesia after either subcutaneous (sc) injections of ketamine (80 mg kg⁻¹ per mouse) and medetomidine (Domitor, Pfizer, 1 mg kg⁻¹ per mouse) or under 5% isoflurane, in order to control for endogenous hormone concentrations and to prevent pregnancies upon repeated testing. At the same time, all females received a 5-mm-long silastic capsule (inner diameter: 1.57 mm; outer diameter: 2.41 mm) containing crystalline 17β-estradiol (diluted 1:1 with cholesterol) subcutaneously in the neck. The dose of E₂ (E8875, Sigma) was based on a previous study^{61 (link)} showing that this treatment leads to estradiol levels similar to mice in estrus. At the end of surgery, females under ketamine/medetomidine anesthesia received an sc injection of atipamezole (Antisedan, Pfizer, 4 mg kg⁻¹ per mouse) to antagonize medetomidine-induced effects and accelerate recovery. In order to induce sexual receptivity at the day of testing, all females received a subcutaneous injection with progesterone (500 µg, P0130, Sigma) 3 h before the onset of the behavioral test, unless stated otherwise (for overview of all the different hormone treatments, see Supplementary Table 1).

In the Cre-LoxP system, activation of Cre recombinase requires 4-hydroxytamoxifen (4-OHT). The 4-OHT was dissolved in DMSO to generate a stock solution at a concentration of 25 mg/mL, and the working concentration was 4 mg/kg diluted in corn oil (C8267, Sigma-Aldrich, St. Louis, MO, USA) at an oil:4-OHT ratio of 9:1. This 4-OHT solution was intraperitoneally injected into E14.5 mice (4 mg/kg/day) along with progesterone (2 mg/day/kg; P0130, Sigma-Aldrich). After birth, 1 mg/kg 4-OHT was injected per day for 5 consecutive days. Then, 2- and 4-week-old mice were collected. Both Ddr1^f/f-4OHT (control group) and OKOΔDdr1 (experimental group) mice were injected with 4-OHT on the abovementioned schedule.