# Melatonin and agomelatine alleviate ivermectin-induced mouse spermatogonia apoptosis via suppression of oxidative stress and calcium overload

## TL;DR

Melatonin and agomelatine protect mouse sperm cells from drug toxicity by reducing oxidative stress and restoring mitochondrial function.

## Contribution

The study identifies structural features of melatonin and agomelatine that are critical for their protective effects against drug-induced mitochondrial damage.

## Key findings

- Melatonin and agomelatine reduce IVM-induced oxidative stress and calcium overload in spermatogonia.
- These compounds restore mitochondrial membrane potential and function, reducing apoptosis and enhancing cell proliferation.
- The methoxy group and N-acetyl side chain are key structural determinants of mitochondrial protection.

## Abstract

Drug toxicity poses a significant threat to male fertility, and its mechanism is often associated with redox imbalance and mitochondrial dysfunction. Ivermectin (IVM), an anthelmintic increasingly explored for new therapeutic applications, induces apoptosis and impairs proliferation in spermatogonia via mitochondria-associated cellular injury at high concentrations in vitro. This study evaluated the protective effects of melatonin, agomelatine, and pinoline, as mitochondria-directed cytoprotectants.

Cultured type B spermatogonia were pretreated with 1 μM melatonin, agomelatine, or pinoline for 24 h under low-serum conditions, followed by exposure to 16 μM IVM. Cell proliferation was assessed by cell counting and Ki67 immunocytochemistry. Mechanistic analyses included fluorescence imaging of reactive oxygen species (ROS) using 2',7'-dichlorodihydrofluorescein diacetate, cytosolic Ca2+ using Fluo-4, AM, and mitochondrial membrane potential (ΔΨm) using tetramethyl rhodamine ethyl ester. Mitochondrial function was evaluated using Seahorse assays, and apoptosis was evaluated by caspase cleavage, the BAX/BCL-2 ratio, and Cytochrome c levels by Western blotting.

Unlike pinoline, melatonin and agomelatine effectively suppressed IVM-induced oxidative stress and Ca2+ overload, while restoring mitochondrial membrane potential (ΔΨm), mitochondrial mass, and oxidative phosphorylation. These protective effects led to reduced apoptosis and enhanced cell proliferation. Structural differences among the three compounds indicate that the methoxy group and N-acetyl side chain are critical determinants of mitochondrial protection under redox stress.

Melatonin and agomelatine protect the male reproductive system from drug-induced toxicity by restoring redox homeostasis and mitochondrial function. These findings provide mechanistic insight into melatonin-based therapeutic strategies and the development of fertility preserving agents targeting mitochondria-mediated cellular injury.