The current study was aimed to evaluate the protective effect of Holothurian atra
(HA) extract; naturally occurring marine resource, against methotrexate (MTX) in-
duced testicular dysfunction. Mature rats received either MTX (20 mg/kg, intraperi-
toneally) or saline on the 7th day of experiment al design. Seven days prior and after
MTX-injection, rats received HA at dose of 300 mg/kg intragastrically (HA + MTX
group; HA group alone). Serum was extracted and testicular tissues were examined
for the changes in serum biochemistry (liver & kidney biomarkers, testicular hor-
mones and antioxidants), molecular and histopthological alterations using RT-PCR
and immunohistochemistry. MTX-injected rats induced alteration in all testicular pa-
rameters. Prior administration of HA ameliorated the MTX-induced oxidative stress.
HA administration normalised MTX-induced decrease in serum levels of interleukin-
6 (IL-6), tumour necrosis factor alpha (TNF-α), interferon-gamma (IFN-γ), reproduc-
tive hormones (FSH, LH and testosterone) and antioxidants GST, SOD and catalase.
MTX-injected rats down-regulated mRNA expression of GST, SOD, steroidogenesis
associated genes, IFN-γ, Bcl2 and NFKB. MTX up-regulated BAX expression and cas-
pase 9 immunoreactivity that were ameliorated in HA + MTX group. Collectively, HA
ameliorated and restored all altered genes. In conclusion, HA is a promising supple-
ment that is helpful in protection against testicular cytotoxicity and dysfunction in-
duced by methotrexate.