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. 2022 May 25;298(7):102077. doi: 10.1016/j.jbc.2022.102077

Figure 2.

Figure 2

Free thiol level and oxidative status are altered in Txnrd3−/−spermatozoa.A, free thiol groups level in epididymal sperm from wildtype and Txnrd3−/− mice. Spermatozoa were isolated from the caput, corpus and cauda epididymis, then stained with ThiolTracker. The intensity indicated the free thiol groups level in sperm. Arrowheads indicate the position of comparison, and empty arrowheads indicate absence/decrease of intensity. B, relative fluorescence intensity (gray value) of free thiol levels in sperm head and midpiece measured by ImageJ. C, immunoblotting analysis of TXNRD3 extracted from caput, corpus, and cauda sperm by urea. D, TXNRD3 is less soluble in SDS in concert with sperm maturation in the epididymis. Acetylated tubulin (AC-TUB) level was used for identical loading evaluation. E, quantification of TXNRD3 expression from D. F, immunoblotting analysis shows GPX4, a potential TXNRD3 substrate, exhibited stronger resistance to lysis buffer in the absence of TXNRD3. Other mitochondrial or midpiece localized proteins such as ANT4 (ADP/ATP translocase 4), P2X2 (P2X purinoceptor 2), and TOM20 (translocase of outer mitochondrial membrane 20) were well solubilized in all detergents tested. GPX4, glutathione peroxidase 4; TXNRD3, thioredoxin-glutathione reductase. Mean ± SD. ∗p < 0.05, ∗∗∗p < 0.001.