In vitro effects of 5-Hydroxy-L-tryptophan supplementation on primary bovine mammary epithelial cell gene expression under thermoneutral or heat shock conditions

Sena L Field,Jimena Laporta,Celeste M Sheftel,Laura L Hernandez,Véronique Ouellet

doi:10.1038/s41598-022-07682-7

Sena L Field, Jimena Laporta + Show 3 more

Open Access

https://doi.org/10.1038/s41598-022-07682-7

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Abstract

Serotonin (5-HT) is an autocrine-paracrine molecule within the mammary gland regulating homeostasis during lactation and triggering involution after milk stasis. Exposure of dairy cows to hyperthermia during the dry period alters mammary gland involution processes leading to reduced subsequent yields. Herein, primary bovine mammary epithelial cells (pBMEC) under thermoneutral (TN, 37 °C) or heat shock (HS, 41.5 °C) conditions were cultured with either 0, 50, 200, or 500 μM 5-Hydroxy-L-tryptophan (5-HTP; 5-HT precursor) for 8-, 12- or 24-h. Expression of 95 genes involved in 5-HT signaling, involution and tight junction regulation were evaluated using a Multiplex RT-qPCR BioMark Dynamic Array Circuit. Different sets of genes were impacted by 5-HTP or temperature, or by their interaction. All 5-HT signaling genes were downregulated after 8-h of HS and then upregulated after 12-h, relative to TN. After 24-h, apoptosis related gene, FASLG, was upregulated by all doses except TN-200 μM 5-HTP, and cell survival gene, FOXO3, was upregulated by HS-50, 200 and 500 μM 5-HTP, suggesting 5-HTP involvement in cell turnover under HS. Supplementing 5-HTP at various concentrations in vitro to pBMEC modulates the expression of genes that might aid in promoting epithelial cell turn-over during involution in dairy cattle under hyperthermia.

Highlights

Serotonin (5-HT) is an autocrine-paracrine molecule within the mammary gland regulating homeostasis during lactation and triggering involution after milk stasis
5-HT is a biogenic monoamine derived from L-tryptophan and converted by tryptophan hydroxylase (TPH1, rate limiting enzyme) to 5-Hydroxy-L-tryptophan (5-HTP, serotonin precursor), which is converted to 5-HT by the aromatic amino acid decarboxylase (AADC, a ubiquitous enzyme). 5-HT can be taken into the cell via the 5-HT reuptake transporter (SERT) and metabolized to 5-Hydroxyindoleacetic acid (5-HIAA)
Consistent with previous reports, there was a potent effect of HS on primary bovine mammary epithelial cells (pBMEC) gene expression, after 8- and 12-h of exposure

Summary

Introduction

Serotonin (5-HT) is an autocrine-paracrine molecule within the mammary gland regulating homeostasis during lactation and triggering involution after milk stasis. The autocrine actions of local factors along with reduced systemic endocrine signals, result in a decline in milk s ynthesis[5] Bioactive factors, such as parathyroid hormonerelated protein, transforming growth factor, insulin-like growth factor and serotonin (5-hydroxytryptamine, 5-HT), accumulate in the milk to elicit microstructure modifications initiating a remodeling of extracellular matrix and cytoskeleton components, as well as a disruption of MEC tight junctions and downregulating milk protein gene expression[6–11]. The efficient removal of senescent MEC to allow the synthesis of new MEC in this redevelopment phase is crucial to maximize milk yield in the lactation[13] Environmental factors, such as heat stress, have been shown to alter this highly regulated cellular process, leading to a less productive mammary gland[13,14]. Exposure of dairy cows to dry period heat stress extends cell death signals beyond involution compromising mammary gland growth prior to parturition and decreasing milk yield in the lactation[15,16]. Intramammary infusions of a selective serotonin reuptake inhibitor, fluoxetine, reduced milk synthesis through an increase in tight junction permeability and increase in plasma lactose concentrations[10]

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