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Serotonin (5-HT) receptor expression in bovine apocrine sweat gland epithelial cells isolated from cow skin
Apocrine sweat glands in bovine skin are involved in thermoregulation. Human, horse and sheep sweat gland epithelial cells have been isolated and grown in vitro. However, isolation of bovine sweat gland epithelial cells (BSGEC) has never been reported. Recent studies have demonstrated that serotonin (5-HT) is an important local regulator of lactational homeostasis and involution in bovine, mouse and human mammary epithelial cells. We hypothesized that since the mammary gland is a modified sweat gland that 5-HT receptors may also be present in BSGEC’s. The present study was conducted to identify a method to isolate bovine sweat glands and culture apocrine BSGECs in vitro and evaluate the expression of 5-HT receptors (1B, 2A, 2B, 4 and 7) in bovine skin, intact apocrine sweat glands and BSGECs.
Collagenase digestion, neutral red staining and mechanical shearing were used to identify and isolate the apocrine glands from skin. The isolated material was transferred to complete media (keratinocyte serum-free media (K-SFM), Bovine Pituitary Extract (BPE) and human recombinant Epidermal Growth Factor (EGF) + 2.5% FBS) in a T25 flask with media film then incubated at 37°C for 24h. After sweat glands adhered to the bottom, an additional 2 ml of complete media was added and the media was changed every 3 days. Isolated apocrine sweat glands and BSGECs were immunostained for cytokeratin and fibroblast specific protein, indicating fibroblast-free cultures. We also determined the mRNA expression of bovine 5-HT receptor subtypes in bovine whole skin, sweat glands and BSGECs. The mRNAs for five receptor isoforms (5-HT 1B, 2A, 2B, 4, and 7) were identified by conventional PCR. We identified isoforms 5HT 1B, 2A, 2B, 4 and 7 in whole skin; 1B, 2B and 7 in isolated sweat glands and BSGECs. We report a method for the isolation of bovine apocrine sweat glands and suggest that keratinocyte medium supplemented with 2.5% FBS is effective and suitable for the culture of BSGECs. The presence of 5-HT receptors in BSGECs indicates that the serotonergic system is involved in regulation of BSGEC function.
Keywords: Apocrine gland, 5-HT receptors, dairy cow