5-Hydroxytryptamine (5-HT) is an amine produced in both the mammary gland and the central nervous system. Tryptophan hydroxylase 1 (TPH1) catalyzes the conversion of 5-hydroxytryptophan (5-HTP) into l-tryptophan, which is then converted into 5-HT by monoamine-oxidase (MAO-A). In the mammary gland, 5-HT has been shown to have a variety of paracrine-autocrine actions, including suppressing lactation, controlling the destiny of mammary epithelial cells, and maintaining calcium homeostasis throughout the transition from pregnancy to lactation. To examine the effects of 5-HT on the composition of colostrum and milk, a total of 30 transition Guan Zhong dairy goats were intramuscularly injected with 5-HTP (1.0 mg/kg) every morning before feeding from 10 d before the projected parturition date to the day of parturition. The average number of days animals received injections was 8.2 ± 3.2 d. 5-HTP treatment increased serum 5-HT concentration from days 5 to 2 relative to parturition (P < 0.05), and decreased the casein concentration of colostrum (P < 0.05). In the in vitro experiment, mammary epithelial cells isolated from three individual goats' mammary glands were separately treated with 200 μM 5-HTP, 30 μM PCPA (the specific inhibitor of TPH1), or 200 μM 5-HTP + 50 μM SB269970 (the selective antagonist of 5-HTR7). The results showed that 200 μM 5-HTP inhibited the expression of β-casein, downregulated the activity of the JAK2/ STAT5a signaling pathway, and promoted the apoptosis of goat mammary epithelial cells (GMECs) (P < 0.05). When GMECs were treated with 30 μM Four-chloro-dl-phenylalanine (PCPA), a specific inhibitor of 5-HT synthesis, the mRNA expression of STAT5a and the phosphorylated STAT5a protein level were upregulated. The 50 μM SB269970 treatment rescued the effects of 5-HTP on GMECs (P < 0.05). Taken together, the results indicated that 5-HTP exerted an inhibitory effect on β-casein synthesis and a proapoptotic effect in GMECs via HTR7 and the JAK2/STAT5a axis.

Background: Calcium is one of the major mineral nutrients in goat milk. Tryptophan hydroxylase1 (TPH1) is a rate-limiting enzyme catalyzing hydroxylation of L-tryptophan into 5-hydroxytryptamine (5-HT) essential for maintaining calcium homeostasis. The function of TPH1 and 5-HT in goat mammary calcium homeostasis is not well known.Methods: The CRISPR/Cas9-meidated TPH1 knockout goat mammary epithelial cells (GMEC) were constructed firstly. Then the content of 5-HT, intracellular calcium level and abundance of key genes related to calcium transportation were evaluated and compared in wild-type GMEC, TPH1 knockout GMEC, to explore the impact of TPH1 on calcium transportation, respectively. Wild-type GMEC and TPH1 knockout GMEC were further treated with exogenous 5-HTP to confirm the role of TPH1 in regulating calcium homeostasis in GMEC. 5-HT concentration was measured by enzyme-linked immunosorbent assay and fluo-3 staining was used to determine intracellular calcium content.Results: The TPH1 knockout GMEC heterozygous clone with no off-target effects was obtained after transfection of the Cas9/sgRNA expression vector. The 5-HT synthesis and intracellular calcium level decreased in TPH1 gene knockout GMEC. The mRNA abundance of secretory-pathway Ca2+ -ATPase1 (SPCA1) and plasma membrane Ca2+-ATPase1 (PMCA1) were up-regulated while the mRNA abundance of secretory-pathway Ca2+ -ATPase2 (SPCA2) was down-regulated in TPH1 knockout GMEC. Up-regulation of parathyroid hormone-related peptide (PTHrP), a key regulator of mammary calcium metabolism, induced by 5-HTP were blocked by TPH1 gene knockout. The TPH1 knockout GMEC showed a lower sensitivity to 5-HTP induced elevation of calcium content.Conclusion: Results suggested that TPH1 plays an important role in regulating calcium homeostasis via PTHrP and calcium transportation related factors in GMEC.

5-羟色胺(5-hydroxytryptamine,5-HT)是一种生物胺和色氨酸的衍生物,可以在动物中枢神经系统和多种外周组织合成,是调节机体生命活动的重要分子.外周组织中合成的5-HT作为一种激素,可以通过自分泌和旁分泌途径影响机体的多种功能.5-HT的受体多样且具有组织和细胞差异性,外周5-HT通过作用于不同的受体参与机体稳态的调节.现综合近年来外周5-HT功能的相关研究,从外周5-HT在不同组织中的调控作用进行综述,旨在为改善母畜泌乳性能、维持骨密度、调节糖脂代谢及控制能量平衡等方面提供思路.

处于围产期的奶山羊由于血液中钙离子迅速降低极易引发低血钙症,5-羟色胺(5-hydorxytyrptamine,5-HT)可以调节生物体内钙代谢,但具体调节机制尚不明确.该研究通过注射5-羟色氨酸(5-HTP,5-hydroxytryptophan)来增加围产期奶山羊体内的5-HT浓度,采集血液提取RNA后,采用全转录组测序技术筛选鉴定差异表达mRNA、microRNA、LncRNA和CircRNA.通过Metascape对差异表达的基因进行富集分析,发现这些差异表达基因主要参与钙代谢、脂质代谢、免疫反应、细胞凋亡等过程.通过预测转录本之间的结合情况构建LncRNA/CircRNA-microRNA-mRNA的竞争内源性RNA (Competing Endogenous RNA,ceRNA)调控网络,并对网络中的基因进行富集分析发现3个与钙代谢相关的基因,分别是Toll样受体9(Toll Like Receptor 9,TLR9)、缝隙连接蛋白γ2 (Gap Junction Protein Gamma 2,GJC2)和连接素1(Junctophilin1,JPH1),LncRNA MSTRG.1354.2可与TLR9竞争性地结合microRNA NC.030825.1_6027,进而调控TLR9的表达,MSTRG.56601.1和MSTRG.101405.3都可以与GJC2、JPH1竞争性地结合NC.030814.1_19917,进而调控GJC2和JPH1的表达.该研究结果为5-HT调控山羊钙代谢提供了一种全新的分子机制.

Serotonin (5-HT) is a monoamine and it could regulate cell growth by its receptors working on signaling pathways. 5-HTP is the precursor of 5-HT that help 5-HT synthesis. B cell leukemia/lymphoma 3 (Bcl-3) involved in cell death and proliferation through mitogen activated protein kinase (MAPK) pathway. However, there is little information about the effects of MAPK/Bcl-3 on apoptosis of goat mammary gland epithelial cells (GMECs). The aim of this study is to explore the interaction among 5-HTP, MAPK and Bcl-3 in GMEC apoptosis. In this study, 5-HTP treatment decreased cell apoptosis and promoted phosphorylation of ERK1/2 in GMEC. We also found that the activation and inhibition of ERK1/2 could affect GMEC apoptosis. The Annexin V-FITC/PI staining and western blotting results suggested that 5-HTP decreased GMEC apoptosis through ERK1/2 signaling pathway. And the results of RT-qPCR and western blotting demonstrated that both 5-HTP and ERK1/2 positively regulated Bcl-3 expression. Sum up all the results, we could draw the conclusion that 5-HTP decreased GMEC apoptosis through MAPK/ERK/Bcl-3 pathway.

5-Hydroxy-l-tryptophan (5-HTP) is the primary product that converts l-tryptophan into 5-hydroxytryptamine by a rate-limiting enzyme. Our previous study found that 5-HTP could promote the intracellular calcium level in goat mammary epithelial cells (GMECs). Herein, first, dairy goats were injected with 5-HTP or saline daily from 7 days before delivery, and the calcium level in colostrum of 5-HTP-injected goats was significantly higher than that of saline-injected goats. Moreover, miR-99a-3p expression was significantly increased after 5-HTP treatment from transcriptome sequencing analysis and quantitative real-time polymerase chain reaction. In addition, it was found that ATP2B1 is one of the target genes of miR-99a-3p predicted by bioinformatic methods, which plays a crucial role in the maintenance of intracellular calcium homeostasis of mammary epithelial cells. Next, we confirmed that miR-99a-3p could increase the intracellular calcium level via decreasing ATP2B1 in GMECs. Taken together, we draw the conclusion that 5-HTP promotes the calcium level in colostrum possibly by increasing intracellular calcium of mammary epithelial cells induced by the miR-99a-3p/ATP2B1 axis.