Progesterone

E3.5 embryos were recovered by flushing uteri with M2 medium. Zona pellucida were removed by brief exposure to acidic Tyrode’s solution (Sigma). Zona-freed blastocysts were seeded on ibiTreat microscopy plastic μ plates (Ibidi), filled with prewarmed IVC1 medium (Advanced DMEM/F12 (GIBCO) containing 20% FCS (Biosera) and supplemented with 2 mM L-glutamine (GIBCO), 1 mM sodium pyruvate (GIBCO), penicillin (25 units/ml)/streptomycin (25 μg/ml) (GIBCO,), 1 × ITS-X (Invitrogen), 8 nM β-estradiol (Sigma), 200 ng/ml progesterone (Sigma), and 25 μM N-acetyl-L-cysteine (Sigma). In the following 36–48 hr, embryos attached to the surface of the plate as TE differentiated into giant cells. The medium was then exchanged and emerging egg cylinders cultured in serum-free, chemically defined IVC2 medium (Advanced DMEM/F12 (GIBCO) containing 30% KSR (KnockOut Serum Replacement, GIBCO) and supplemented with 2 mM L-glutamine (GIBCO), 1 mM sodium pyruvate (GIBCO), penicillin (25 units/ml)/streptomycin (25 μg/ml) (GIBCO), 1 × ITS-X (Invitrogen), 8 nM β-estradiol (Sigma), 200 ng/ml progesterone (Sigma), and 25 μM N-acetyl-L-cysteine (Sigma). Embryo culture was performed at 37°C in 5% CO₂. Procedures used for imaging living or fixed preparations of cultured embryos are given in the Extended Experimental Procedures.

For genetic cell labelling we crossed tamoxifen-inducible Csf1r^MCM, Tie2^MCM, and Kit^MCM with Rosa26^eYFP, Rosa26^GFP, or Rosa^mTmG reporter mice. In Csf1r^MCM embryos recombination was induced by single injection at E8.5 of 75 µg per g (body weight) of 4-hydroxytamoxifen (Sigma) into pregnant females. The 4-hydroxytamoxifen was supplemented with 37.5 µg per g (body weight) progesterone (Sigma) to counteract the mixed oestrogen agonist effects of tamoxifen, which can result in fetal abortions. In Tie2^MCMRosa26^eYFP embryos recombination was induced by treatment of pregnant females by gavage at different timepoints at E7.5 and E10.5 with a single dose of 2.5 mg tamoxifen (Sigma) and 1.25 mg progesterone. In Kit^MCM animals recombination was induced in 6–8-week-old adult animals using oral tamoxifen feeding (400 mg/kg diet, which approximates to a daily dose of 40 mg/kg mouse body weight; purchased from Envigo).

All experimental procedures involving mice followed a protocol approved by the Washington University in St. Louis Institutional Animal Care and Use Committee (Protocol Number 20160227). CD1 wild-type mice (Charles River, Saint Louis, MO, United States) were maintained on a 12-h light:12-h dark cycle. To assess uterine progesterone responses, 6-week-old CD1 mice were bilaterally ovariectomized, allowed to rest for 2 weeks to allow the endogenous ovarian-derived steroid hormones to dissipate, and then subcutaneously injected with 100 μl of sesame oil (vehicle control) or 1 mg of progesterone (Sigma-Aldrich) in 100 μl of sesame oil. Six hours later, mice were euthanized, uterine tissues were collected, and RNA was isolated and processed for qRT-PCR.

To label Mrgprd⁺ nociceptors, Mrgprd^CreERT2 mice carrying the relevant reporter allele were treated with tamoxifen ((Sigma-Aldrich, St. Louis, MO, T5648) pre- or postnatally. For prenatal treatment, pregnant females were given tamoxifen along with estradiol (Sigma, E8875, at a 1:1000 mass estradiol: mass tamoxifen ratio) and progesterone (Sigma, P3972, at a 1:2 mass progesterone: mass tamoxifen ratio) in sunflower seed oil via oral gavage at E16.5-E17.5, when Mrgprd is highly expressed in mouse non-peptidergic nociceptors (Chen et al., 2006 (link)). For postnatal treatment, 0.5 mg tamoxifen extracted in sunflower seed oil was given via i.p. injection once per day from P10-P17 (or P14-P21 for spinal cord slice recording experiments, Figure 7). At least one week was given to drive recombination and reporter gene expression.

This experiment was designed to determine the hormonal effect on EVs produced by endometrial cells. RL95-2 cells were cultured in T75 flasks with complete DMEM culture media until ~60/70% confluent. The media was changed after being washed using DPBS without Ca²⁺ and Mg²⁺ 3 times before being incubated with phenol red free DMEM culture media with charcoal stripped FBS (10%) to adapt similar conditions and avoid possible hormone-like (estrogenic) activity of phenol red for 24 h. The appropriate hormone treatment was then added to the cells in phenol red-free DMEM culture media at either a final concentration of 10 nM of progesterone (water soluble, Sigma-Aldrich, Dorset, UK) (P4) or 10 nM of estrogen (water soluble, Sigma-Aldrich, Dorset, UK) (E2) or estrogen (10 nM) plus progesterone (100 nM) (E2P4), as described before [18 (link),19 (link)]. A control group containing equal amounts of the solubility complexing agent, cyclodextrin, found within the hormone mixtures was also performed. Following a 24 h incubation period, conditioned media was collected, and differential centrifugation was performed, followed by concentration and size exclusion chromatography, as described above. Samples were then analysed for particle concentration via NTA and protein concentration using the BCA assay. Experiments were performed in triplicates on 3 different days.