Evaluating the reproductive toxicity of antidepressants is particularly important for those depressed male patients of childbearing age and crucial for the rational use of drugs in clini

Huang, Gelin

Li, Peiyu

Li, Tongtong

Li, Xiaoliang

Shang, Lijun

Wu, Sixian

Xu, Wenming

London Met Repository

Developing testicular organoids for assessing reproductive toxicity of antidepression drugsSixian Wu a, †, Xiaoliang Li a, b, †, Peiyu Li a, Tongtong Li a , Gelin Huang a, Lijun Shang c, * and Wenming Xu a, *a. Joint Laboratory of Reproductive Medicine, SCU-CUHK, Key Laboratory of Obstetric, Gynaecologic and Paediatric Diseases and Birth Defects of Ministry ofEducation, West China School of Medicine, West China Second University Hospital, Sichuan University, Chengdu 610041, P. R. China. b. Reproductive Centre,Department of Obstetrics and Gynaecology, West China Second University Hospital, Sichuan University, Chengdu, China. c. School of Human Sciences, LondonMetropolitan University, London, N7 8DB, UK. † Equal contribution * Co-corresponding authorsReferences1. A. F. Ajayi and R. E. Akhigbe, Andrologia, 2020, 52, e13672.2. 2. S. Almeida, L. et al, Current pharmaceutical design, 2017, 23, 4429-4437.3. P. C. Barrow, Reprod Toxicol, 2009, 28, 172-179.4. L. A. Burns-Naas, et al, Regulatory Toxicology and Pharmacology, 2003, 38, 304-316.5. J. P. Alves-Lopes, et al, Nat Protoc, 2018, 13, 248-259.6. J. Clinton and P. McWilliams-Koeppen, Curr Protoc Cell Biol, 2019, 82, e66.Introduction Evaluating the reproductive toxicity of antidepressants is particularly important for those depressed male patients of childbearingage and crucial for the rational use of drugs in clinic [1,2]. Traditional methods are unable to precisely discern how antidepressants affect sperm[3,4]. In this study, we developed an improved testicular organoid [5] which had all cells (germ cells, spermatocytes, supporting cells) related tosperm production and developed the blood testicular barrier to maintain the testicular microenvironment. Most importantly, this improved testicularorganoid could prolong the life span. Using this improved testicular organoid, we evaluated in detail the effects of two most clinical usedantidepression drugs, amitriptyline and mirtazapine on sperm development.Aim and methods We introduced a new 3-layer gradient system (LGS) by changing the testicular organoid culture conditions such as thenumber of cells and the concentration of Matrigel. By doing so, the area of cytokine exchange around the organoid was greatly increased. Thisprovided more nutrients for the growth and the culture of the organoid. These changes consequently facilitated a long-term culture of theorganoid. We next used this improved testicular organoid to assess the reproductive toxicity of antidepression drugs, in particularly to mimic thelong-term effect of the drugs on sperm. Two most used clinical drugs, amitriptyline and mirtazapine, were chosen to directly observe their effectson spermatogenic cells so that their reproductive toxicities can be evaluated.ResultsConclusion The testicular organoids developed in this study can mimic thesperm development and then be successfully used to assess thereproductive toxicity. It can sensitively discern the changes between differentdrugs and their effects on the spermatogenesis process. This provides apowerful tool to observe more comprehensive dynamic process ofspermatogenesis and to analyze key signal pathways affected by drugs onspecific type of cells. This feasible solution for the evaluation of reproductivetoxicity in the drug development certainly will attract further expedition todevelop human testicular organoid and of course provide insight knowledgeof reproductive toxicity of clinical drugs.Figure 2. The characterization of organoids. Immunostaining images of Ddx4 (green), Sox9 (red), Zo1 (red) and Scp3 (green) genes for 7-day cultured organoid.  Scale bars = 50 µm.Figure 3. (A) Staining images of Hoechst (Blue)& PI (Red) after treating organoids with 1 μM and 100 nM amitriptyline and mirtazapine for 96 h. (B) The fluorescence ratio statistics chart, R=red/blue=PI/Hoechst. R value was less than 0.21 for the organoids treated with mirtazapine while greater than 0.21 for those organoids treated with amitriptyline.Figure 1. The testicular organoid model. The model has 3-layer gradient system with careful selection of cell culture conditions shown in the figure. A layer of 5 μL Matrigel is on the support. 1 μL volume of cell suspension (medium: Matrigel=1:1) is on the Matrigel. The outermost layer is wrapped by 9-10 μL Matrigel.Figure 4. The mRNA expression of Zo1, Sox9, Stra8 and Uchl1 after treatment with 1 μM and 100 nM mirtazapine and amitriptyline for 48 h and 96 h. JQ1 was used a positive control. Gapdh is used as an internal reference gene for qPCR. The number of animals in each group n=3, *p<0.05, **p<0.01.Figure 5. A schematic diagram of the blockade of spermatogenesis with amitriptyline and mirtazapine. “↓” means down regulation, “↑” means up regulation, “-” means no change, “↓ (purple)” means the degree to which mirtazapine affects spermatogenesis, “↓ (red)” means the degree to which amitriptyline affects spermatogenesis, “↓ (purple/red)” the more Indicates that mirtazapine/amitriptyline has a greater impact on spermatogenesis.

Developing testicular organoids for assessing reproductive toxicity of antidepression drugs

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Developing testicular organoids for assessing reproductive toxicity of antidepression drugs

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