Primary Cell Culture

Isolation and culture of primary mouse brain microvascular endothelial cells (pMBMECs) from 7–12‐week‐old female mice were performed as previously described (Coisne et al, 2005 (link)). Briefly, mice were euthanized by cervical dislocation, brains were dissected, and meninges, olfactory bulb, brainstem, and thalami were removed. A minimum of six brains per genotype were pooled, homogenized, and resuspended in a 30% dextran (Sigma) solution to obtain a final concentration of 15% dextran. Samples were centrifuged, the vascular pellet was collected, and then digested by incubation with Collagenase/Dispase (2 mg/ml), DNAse I (10 μg/ml) and Nα‐Tosyl‐L‐Lysin‐chlormethyl keton hydrochlorid (TLCK, 0.147 μg/ml) for 30 min at 37°C in a shaker incubator. Digested vessel fragments were then plated in Matrigel (Corning)‐coated 96‐well Nunclon Delta Surface (Thermo Scientific) plates at a seeding density of 51,000 digested capillaries/cm² onto Matrigel‐coated wells. pMBMECs were maintained in Dubelco's Modified Eagle Medium (DMEM, Gibco) with 10% FCS (Biowest), 50 μg/ml of gentamicin (Sigma), and 1 ng/ml of basic fibroblast growth factor (Sigma) at 37°C and 5% CO₂ in a humidified incubator. Once confluent, cells were treated with either mouse recombinant IL‐1β (R&D systems, 10 ng/ml) or vehicle for 24 h.

Bovine ovaries from randomly cycling cattle and other endocrine tissues (pituitary glands, testes and adrenal glands) were obtained from an abattoir. Ovaries were dissected to obtain antral follicles ranging in diameter from 3 mm to 18 mm, and these were further processed to isolate the GC layer, TC layer, and follicular fluid, as described previously (Glister et al. 2010 (link)). Briefly, follicles were sorted into five different size classes: 3–4 mm (n = 8), 5–6 mm (n = 8), 7–8 mm (n = 9), 9–10 mm (n = 6) and 11–18 mm (n = 12). Each follicle was hemisected, and GC and TC layers were recovered for RNA extraction, while follicular fluid was recovered for steroid hormone analysis. Large follicles (11–18 mm) were reclassified according to their oestrogen to progesterone ratio (E:P ratio) in follicular fluid as either large oestrogen-active (LEA; E2:P4 ratio >1) or large oestrogen-inactive (E2:P4 ratio <1) follicles. Corpora lutea (CL) at growing (n = 4), mid-luteal (n = 5) and regressing (n = 4) stages were also harvested. All tissue samples were homogenized in Trizol reagent for total RNA extraction, as described previously (Glister et al. 2010 (link)).
Follicles (4–8 mm diameter) were also retrieved for isolation of GC and TC to be used for primary cell culture experiments, as described in detail elsewhere (Glister et al. 2001 (link), 2005 (link)). GC and TC were seeded into 96-well plates (Nunclon, Life technologies Ltd.) at a density of 75,000 cells/250μL/well for serum-free culture (non-luteinized cells) or 10,000 cells/250 μL/well for serum-supplemented culture (luteinized cells). Cells were cultured for 6 days at 38.5°C with saturating humidity in 5% CO₂ in air. The culture medium consisted of McCoy’s 5A medium (Sigma), supplemented with antibiotic/antimycotic solution (1% v/v; Sigma), apo-transferrin (5 μg/mL; Sigma), sodium selenite (5 ng/mL; Sigma), bovine insulin (10 ng/mL; Sigma), HEPES (20 mM; Sigma) and bovine serum albumin (0.1% w/v; Sigma). Medium used for serum-free GC culture was also supplemented with 10^–7 M androstenedione as aromatase substrate. For GC and TC cultured under conditions that promote luteinization, 2% fetal calf serum (FCS) was also included as a supplement. In all four culture models, media were changed after 48 h and replaced with fresh media containing treatments as specified below. This was repeated after a further 48-h incubation period. Media were retained after the final 48-h period (i.e. 96–144 h) for subsequent analysis of steroid hormone secretion. Viable cell number at the end of culture was determined by neutral red uptake, as described elsewhere (Glister et al. 2001 (link)).
It should be noted that culturing TC and GC using defined serum-free medium preserves a non-luteinized phenotype reflected by LH-induced androstenedione (A4) secretion by TC and follicle-stimulating hormone (FSH)-induced oestradiol (E2) secretion by GC. Henceforth, these cells will be referred to as non-luteinized TC (NLTC) and non-luteinized GC (NLGC). In contrast, culturing TCs and GCs under serum-supplemented conditions promotes spontaneous luteinization, as indicated by reduced A4/E2 secretion and greatly increased secretion of P4 (Glister et al. 2001 (link), 2005 (link), Kayani et al. 2009 (link)). Henceforth, these cells will be referred to as LTC and LGC.