1st Research Ameliorative effect of combined low dose of Pioglitazone and omega-3 on spermatogenesis and steroidogenesis in diabetic rats Mai M. Hasan1 | Amal S. El-Shal2 | Amal M. H. Mackawy2, 3 | Ebtesam M. Ibrahim1 | Eman M. M. A. Abdelghany4 | Abeer A. Saeed1 | Jehan El-Gendy5 1Department of Medical Physiology, Faculty of Medicine, Zagazig University, Zagazig, Egypt. 2Department of Medical Biochemistry, Faculty of Medicine, Zagazig University, Zagazig, Egypt. 3Department of Medical Lab, Applied Medical Science, Qassim University, Qassim, KSA 4Department with Anatomy and Embryology, Faculty of Medicine, Zagazig University, Zagazig, Egypt. 5Department with Medical Pharmacology, Faculty of Medicine, Zagazig University, Zagazig, Egypt. J Cell Biochem. 2020;121:1524–1540. . https://doi.org/10.1002/jcb.29388 Abstract Background: Chronic hyperglycemia is linked to either subfertility or infertility among diabetic males. Pioglitazone is one of the thiazolidinediones (TZDs) drugs that are selective peroxisome proliferator-activated receptor (PPAR-γ agonists used for treating type 2 diabetes mellitus (T2DM). Aim: This study aims to explore the possible effect of low Pioglitazone dose and omega (ω-3) on rat male reproductive function. Furthermore, we evaluated the add-on effect of combined use of low Pioglitazone dose of and ω-3 on reproductive functions in adult male T2DM rats. Methods: Fifty adult male rats were included and subdivided into control and four test subgroups. T2DM was induced in test groups and subdivided into non-treated T2DM, ω-3 treated, 0.6mg/kg Pioglitazone treated, and combined treated group (orally by gavage). Following 16 weeks, final body weight, testicular weight, fasting plasma glucose, and serum testosterone levels were measured. Semen analysis, testicular testosterone, malondialdehyde (MDA) concentrations, superoxide dismutase (SOD) activity, immunohistochemistry staining for apoptosis marker B-cell lymphoma protein 2 (Bcl-2), proliferation marker as proliferating cell nuclear antigen (PCNA), estrogen receptor α (ERα), androgen receptor (AR) were determined. Caspase-3, nuclear factor-kappa B (NF-kB), glucose transporter 3 (GLUT3), 17β-hydroxysteroid dehydrogenases (17β-HSD) PPARγ, and PPARα genes expression were analyzed by real-time polymerase chain reaction (RT-PCR). Results: Our findings revealed that treatment with low dose of Pioglitazone or ω-3 significantly lowered fasting plasma glucose and MDA levels, ameliorated diabetes effects on histological damage, improved antioxidant activity (SOD), significantly improved anti-apoptosis BCL-2 and proliferation (PCNA), remarkably elevated ERα, AR, 17β-HSD PPARγ, and PPARα expression with significant reduction in caspase-3, NF-kB genes expression and improved semen quality as well. Combined use of low dose of and ω-3 has better effects on all measured parameters. Conclusion: Small Pioglitazone dose and ω-3 possess beneficial effects on spermatogenic and steroidogenic functions in adult diabetic rat; while combined use of both has an add-on effect. Keywords: Pioglitazone, gene expression, rat male fertility, hyperglycemia, omega-3, PPAR, real-time polymerase chain reaction