Tabriz University of Medical Sciences Advanced Pharmaceutical Bulletin 2228-5881 16 3 2026 09 30 Autophagy Modulation Influenced the Spermatogenesis in Rodent Testicular Organoids and Diabetic Condition 593 605 10.34172/apb.46240 EN Shadi Atazadeh https://orcid.org/0009-0001-6767-796X Maryam Hasan Sadeh https://orcid.org/0009-0006-7489-802X Farah Farokhi https://orcid.org/0009-0002-9859-9965 Shahin Ahmadian https://orcid.org/0000-0003-1443-278X Sepideh Saghati https://orcid.org/0000-0002-2801-3482 Fereshteh Valipour https://orcid.org/0000-0002-4126-3945 Amir Fattahi https://orcid.org/0000-0002-5712-5336 Amin Tamadon https://orcid.org/0000-0002-0222-3035 Reza Rahbarghazi https://orcid.org/0000-0003-3864-9166 Mahdi Mahdipour https://orcid.org/0000-0002-2729-4593 Journal Article 10.34172/apb.46240 2025 08 17 2026 06 22 Introduction: Here, the effect of autophagy activity was investigated in rat testicular organoids embedded in alginate/gelatin (Alg/Gel) hydrogel. Additionally, we monitored the spermatogenesis potential in diabetic mice following the administration of metformin (Met) and/or hydroxychloroquine (HCQ). Methods: Alg/Gel hydrogel was fabricated, and its physicochemical properties were studied. The survival rate of freshly isolated rat whole testicular tissue cells being plated on the Alg/Gel hydrogel surface was monitored after 7 days. Rat testicular organoids were embedded in Alg/Gel hydrogel and exposed to 15 µM HCQ and/or 5 µM Met for 7 days. Histological examination was conducted to assess OCT3/4+ progenitors and maturing sperm cells. Using western blotting, the levels of autophagic markers Beclin-1, LC3, and P62 were evaluated. Diabetic mice received 120 mg/kg body weight Met and/or 70 mg/kg bodyweight HCQ for 4 weeks, followed by histological examinations and sperm analysis. Results: The fabricated Alg/Gel hydrogel demonstrated suitable physicochemical properties, which correlated with an increased survival rate of whole testicular tissue cells compared to the control group (P<0.05). Data indicated a promotion of autophagy (Beclin-1↑, LC3-II/-I ratio↑, P62↓) and an inhibition of autophagy (LC3↓) in testicular organoids after treatment with Met and HCQ, respectively, compared to the control group (P<0.05). These changes were accompanied by an induction of SOD activity in Met-treated testicular organoids (P<0.05). In the presence of Met and HCQ, OCT3/4+ progenitors were reduced in rat organoids compared to non-treated control organoids (P<0.05). Administration of Met, but not HCQ, in diabetic mice was able to restore some sperm parameters, such as motility, and reduce abnormal morphologies compared to the matched diabetic mice (P<0.05). Conclusion: Regulation of autophagy can influence spermatogenesis capacity, affecting seminiferous tubule progenitors, maturation, and sperm parameters. In studies of rat testicular organoids and diabetic mice, these findings suggest that the autophagy response is a promising signaling pathway target in subfertile or infertile males.