Decoding a novel non-enzymatic protein acetylation mechanism in sperm that is essential for fertilizing potential

Abstract

Background: Protein acetylation has emerged as essential for sperm function, attracting considerable attention recently. Acetylation, typically mediated by lysine acetyltransferases, involves attaching an acetyl group from acetyl-coenzyme A to lysine residues in proteins. Under alkaline conditions, however, acetylation can occur with minimal enzymatic involvement, primarily due to an elevated pH. As sperm migrate towards the ampulla, they experience increasing intracellular pH (pHi) while undergoing two crucial processes for fertilization: capacitation and the acrosome reaction (AR). Whereas the involvement of acetylating enzymes in these events has been partially investigated, the potential for non-enzymatic acetylation driven by the pHi alkalinization remains unknown.

Results: This study examined protein acetylation (acLys) levels in sperm incubated under capacitating conditions at pH 7.2 and pH 9.0, the latter condition potentially promoting non-enzymatic acetylation. To more precisely investigate the occurrence of non-enzymatic acetylation events, acetyltransferase activity was selectively attenuated using a specific cocktail of inhibitors. The functional implications of these conditions were assessed by examining key fertilization-related sperm attributes, including motility during capacitation and the ability to initiate the AR. Results demonstrated that alkaline conditions elevated basal acLys levels even with reduced acetyltransferase activity (P < 0.05), indicative of non-enzymatic acetylation. α-tubulin, particularly in the midpiece of the sperm flagellum, was identified as a specific target of this modification, correlating with diminished motility during capacitation. Following the AR, acLys levels in the head and midpiece decreased (P < 0.05) under conditions promoting non-enzymatic acetylation, accompanied by reductions in intracellular and acrosomal pH. In contrast, acLys levels and pH in the sperm head incubated under standard capacitating conditions (pH 7.2) remained stable. Sperm exposed to conditions conducive to non-enzymatic acetylation exhibited an impaired ability to trigger the AR (P < 0.05) compared to those maintained at pH 7.2. Notably, diminished acetylase activity emerged as a key factor impairing the maintenance of intracellular and acrosomal pH levels attained during capacitation, even under a pH of 9.0.

Conclusion: This study provides novel evidence for the occurrence of non-enzymatic acetylation in sperm, linked to the modulation of α-tubulin acetylation levels and motility during capacitation. Additionally, it suggests that acetyltransferase activity may play a crucial role in regulating intracellular and acrosomal pH levels in capacitated sperm, facilitating the AR.

Keywords: Acrosome reaction; Motility; Non-enzymatic acetylation; Sperm capacitation; pH; α-tubulin.

Fig. 1

Capacitation achievement, interplay between acetylases activity and extracellular pH (pHe), and evidence of non-enzymatic acetylation events in sperm. Sperm were incubated under non-capacitating (NCAP) and capacitating (CAP) conditions, as well as under capacitating conditions with the acetylase inhibitor cocktail (Cocktail) at pH 7.2 (white bars) and 9.0 (grey bars), and A sperm motility, B intracellular pH (pHi), and phosphorylation of C protein kinase A (pPKA) and D tyrosine residues (pY) were evaluated. Immunoblotting with anti-pPKA substrates and anti-pY antibodies revealed prominent 42 and 32 kDa protein bands, respectively, in sperm extracts incubated under standard capacitating conditions at pH 7.2 (arrowheads). Complementary IF results are shown, including E the histogram of Live/Dead™ Fixable violet dye (L/D) for the differentiation of the viable sperm population and the quantification of F pPKA and G pY in that specific population. Acetylation levels of sperm expressed as percentages of sperm with acetylated proteins in the H head, K midpiece, and N principal piece, and as accumulation of acetylated proteins (acLys) in these regions (I, L, O, respectively). Representative images of acetylated proteins in the J head, M midpiece, and P principal piece from the flow cytometry AMNIS image unit are provided. The accumulation of F pPKA, G pY and I, L, O acetylated proteins is expressed as the fold change in mean fluorescence intensity (MFI) relative to the NCAP at pH 7.2 and 9.0 (assigned a value of 1; RMFI). All columns show mean ± s.e.m. (* P < 0.05, ** P < 0.01). The Wilcoxon signed-rank test and one-sample t-test were employed to compare the mean of pHi after incubation with the baseline pHi at the onset of the experiment (i.e. ± 7.2; P = 0.05). Scale bar = 20 µm

Fig. 2

α-Tubulin is targeted by non-enzymatic acetylation, and its accumulation in the midpiece of the flagellum is associated with decreased sperm motility. A Immunoblotting with anti-acetylated α-tubulin (acTub) and B integrated density (IntDen) of bands signals following incubation under non-capacitating (NCAP) and capacitating (CAP) conditions, as well as under capacitating conditions with the acetylase inhibitor cocktail (Cocktail) at pH 7.2 (white bars) and 9.0 (grey bars). For additional localization details of acTub, immunofluorescence was performed and analyzed using flow cytometry, and the results of the sperm percentage undergoing acTub and its accumulation are shown for the C, D head, F, G midpiece and I, J principal piece of the tail. Representative images of acetylated proteins in the E head, H midpiece, and J principal piece from the flow cytometry AMNIS image unit are provided. The accumulation of acTub is expressed as the fold change in mean fluorescence intensity (MFI) relative to the NCAP sample at each pH (assigned a value of 1; RMFI). Percentage of sperm comprising subpopulations K I and L II (SPI and SPII) after application of principal component analysis and clustering of sperm based on their kinetics characteristics after capacitation. All columns show mean ± s.e.m. (* P < 0.05). Scale bar = 20 µm

Fig. 3

Evaluation of protein acetylation after inducing the AR. Following incubation in non-capacitating (NCAP) and capacitating (CAP) conditions, as well as under capacitating conditions with the acetylase inhibitor cocktail (Cocktail) at pH 7.2 (white bars) and 9.0 (grey bars), sperm were exposed to progesterone (10 µM/mL–10 min) to induce the AR. First, acrosome integrity was evaluated to corroborate the occurrence of the AR in the conditions under study. A Percentages of viable sperm exhibiting an intact acrosome assessed by FITC-PNA staining and flow cytometry. Acetylation levels in sperm expressed as percentage of viable sperm with acetylated proteins in the B head, C midpiece, and D principal piece, and as accumulation of acetylated proteins in these regions (E, F, G, respectively). The accumulation of acetylated proteins is expressed as the fold change in mean fluorescence intensity (MFI) relative to the NCAP sample at each pH (assigned a value of 1; RMFI). Intracellular and acrosomal pH of sperm incubated in the conditions under study after capacitation (H, K, respectively), and the AR (I, J, respectively). As alkalinization of acrosomal pH led to a decrease in Lysosensor™, the acrosomal pH is expressed as the fold change in mean fluorescence intensity (MFI) of the Lysosensor™ fluorochrome relative to the NCAP sample at each pH (assigned a value of 1; RMFI). Representative histograms of L BCECF-AM and M Lysosensor™ intensities assessed by flow cytometry. BCECF-AM fluorescence shifted from 525 to 640 nm in a pH-dependent manner; thus, BCECF-AM intensity is expressed as 525/640 nm ratio. Incubation in CAP at pH 7.2 resulted in the appearance of a sperm subpopulation with a more alkaline pH compared to those incubated in NC at pH 7.2. This subpopulation exhibited a higher 525/640 nm ratio, appearing to the right of the main population. As Lysosensor™ labels acidic organelles such as the acrosome, a decrease in its intensity reflects alkalinization. All columns show mean ± s.e.m. (* P < 0.05)

Fig. 4

Differences of acetylated proteins (acLys), including acTub prior and post-AR of sperm incubated in capacitating (CAP) conditions, between the presence and the absence of the acetylase inhibitor cocktail (Cocktail) at pH 7.2 (white bars) and 9.0 (grey bars). For AR induction, after 3 h of incubation in the indicated conditions, sperm were exposed to progesterone (10 µM/mL) for further 10 min. Acetylation levels before and after the AR are shown for the A head, B midpiece, and C principal piece. Due to the observed reduction in acLys, acTub was assumed to be a candidate protein experiencing modifications post-AR. Then, acTub levels were evaluated and expressed as the percentage of sperm with acTub, and accumulation of acTub in the D, E head, F, G midpiece, and H, I principal piece. Differences of accumulated acTub before and after AR induction in the J head, K midpiece, and L principal piece. The accumulation of acTub is expressed as the fold change in mean fluorescence intensity (MFI) relative to the NCAP sample at each pH (assigned a value of 1; RMFI). Differences in M intracellular and N acrosomal pH after the AR and O reduction in acrosome integrity of sperm incubated under the specific conditions after inducing the AR. All columns show mean ± s.e.m. (* P<0.05)