Modulation of nicotinamide adenine dinucleotide phosphate oxidase activity through sequential posttranslational modifications of p22 phagocytic oxidase during capacitation and acrosome reaction in goat spermatozoa1

Abstract

Superoxide anion radical, produced in low quantities, plays a positive role in sperm function. Spermatozoa produce superoxide anion radical during posttesticular development, which shows an abrupt increase during capacitation. The NAD phosphate oxidase (NOX) family members NOX2 and NOX5 are the 2 enzymes implicated in superoxide production in spermatozoa. We examined the organization of NOX2 in goat spermatozoa during epididymal maturation, capacitation, and acrosome reaction. Spermatozoa from testis, caput epididymidis, corpus epididymidis, and cauda epididymidis possessed components of the phagocytic oxidase (PHOX; i.e., gp91phox, p22phox, p67phox, p47phox, p40phox), and ras-related C3 botulinum toxin substrate 1/2 (Rac1/2) on spermatozoa, and their concentrations did not show significant alterations during epididymal maturation. During capacitation in vitro, p22phox underwent Thr-phosphorylation, which resulted in a mobility shift of the corresponding band toward greater molecular mass. The Rac1/2 also showed a mobility shift from 32 to 23 kDa during capacitation. During progesterone-induced acrosome reaction, the spermatozoa experienced a total loss of p22phox and p47phox. The p47phox, but not p22phox, was detected in the exocytic vesicles of the acrosome. The Thr-phosphorylated form of p22phox was ubiquitinated and degraded through proteasome-mediated pathways in goat sperm cell lysates. Thus, Thr phosphorylation of p22phox acts as a regulatory switch in goat spermatozoa that transiently activates the NOX2 system during capacitation and subsequently directs it for degradation through the ubiiquitin-proteasomal pathway during progesterone-induced acrosome reaction.

Reciprocal regulation between sirtuin-1 and angiotensin-II in the substantia nigra: implications for aging and neurodegeneration

“…The membranes were incubated overnight with primary antibodies rabbit anti-SIRT1 (Millipore 07-131), goat anti-AT1 (sc-31181), rabbit anti-AT2 (sc-9040), and rabbit anti-p47 phox (sc-14015) from Santa Cruz Laboratories 1:200. The specificity of the antibodies was established in previous studies: AT1 sc-31181 [ 71 ], AT2 sc-9040 [ 72 , 73 ], p47 phox sc-14015 [ 74 ], Sirt1 Millipore 07-131[ 75 , 76 ]. In addition, the specificity of the antibodies was confirmed in our laboratory by preadsortion with the corresponding synthetic peptide antigen [ 77 ] and western blot analysis of lysates from HEK293 cells transfected with AT1 or AT2 tagged to fusion tail DDK (TA50011 from Origene; DDK tag: DYKDDDDK).…”

Research on early mammalian development in India

“…Besides, work was also carried out on screening of Indian plants for antifertility effect (Satyavati, 1984) and on endocrine disruptors (Batra, 1966). During later part of the last century and the early part of this century, India contributed to the area of spermatogenesis and sperm function involving the molecular regulation of male germ (stem) cell development and differentiation (Mitra and Shivaji, 2004;Seshagiri et al, 2007;Parab et al, 2015;Dumasia et al, 2016) and the ontogeny of differentiation-associated signalling systems during germ cell development (Chandrasekhar et al, 2011;Bhattacharya et al, 2012;Majumdar et al, 2012).…”

“…Using rodent models and the human, Indian scientists contributed to the understanding of the capacitation-associated changes in sperm protein phosphorylation and their functional significance in sperm hyper-activation phenomenon (Ain et al, 1995(Ain et al, , 1999Mitra and Shivaji, 2004;Mariappa et al, 2006;Seshagiri et al, 2007;Mariappa et al, 2010;Chandrasekhar et al, 2011;. Besides, the functional significance of progesterone and HSP90 involved in sperm hyper-activation was determined and the molecular causes of male factor infertility (Majumdar et al, 2012;Basu et al, 2018).…”

Human sperm Toll-like receptor 4 (TLR4) mediates acrosome reaction, oxidative stress markers, and sperm parameters in response to bacterial lipopolysaccharide in infertile men

“…Sperm protein extraction was performed following a previously described method [30]. Briefly, for each sperm sample, 2 × 10 6 of selected human spermatozoa were twice washed with 500 μl of phosphate-buffered saline (PBS; pH 7.4) and centrifuged at 10,000×g for 10 min.…”