Leydig cells are responsible for
testosterone production in male testis upon stimulation by
luteinizing hormone.
Inflammation and oxidative stress related Leydig cell dysfunction is one of the major causes of
male infertility.
Cytoglobin (CYGB) and
Neuroglobin (NGB) are two
globin family member
proteins which protect cells against oxidative stress. In the current study, we established a
Lipopolysaccharide (LPS)-induced
inflammation model in TM3 Leydig cell culture to study the function of CYGB and NGB
proteins under inflammatory conditions. CYGB and NGB were downregulated using
siRNA and
shRNA based experimental strategies. Overexpression was conducted using lentiviral pLenti-III-CYGB-2A-GFP, and pLenti-III-NGB-2A-GFP vector systems. As testicular macrophages regulate immune function upon
inflammation and steroidogenesis of Leydig cells, we generated direct/indirect co-culture systems of TM3 and mouse macrophage (RAW264.7) cells ex vivo. Downregulation of CYGB and NGB induced nitride
oxide (NO) release, blocked cell cycle progression, reduced
testosterone production and increased inflammatory and apoptotic pathway gene expression in the presence and absence of LPS. On the other hand, CYGB and NGB overexpression reduced TNFα and COX-2
protein expressions and increased the expression of
testosterone biogenesis pathway genes upon LPS stimulation. In addition, CYGB and NGB overexpression upregulated
testosterone production. The present study successfully established an inflammatory interaction model of TM3 and RAW264.7 cells. Suppression of CYGB and NGB in TM3 cells changed macrophage morphology, enhanced macrophage cell number and NO release in co-culture experiments upon LPS exposure. In summary, these results demonstrate that
globin family members might control LPS induced
inflammation by regulating apoptotic mechanisms and macrophage response.