Adenine nucleotide metabolism and a role for AMP in modulating flagellar waveforms in mouse sperm

Abstract

While most ATP, the main energy source driving sperm motility, is derived from glycolysis and oxidative phosphorylation, the metabolic demands of the cell require the efficient use of power stored in high-energy phosphate bonds. In times of high energy consumption, adenylate kinase (AK) scavenges one ATP molecule by transphosphorylation of two molecules of ADP, simultaneously yielding one molecule of AMP as a by-product. Either ATP or ADP supported motility of detergent-modeled cauda epididymal mouse sperm, indicating that flagellar AKs are functional. However, the ensuing flagellar waveforms fueled by ATP or ADP were qualitatively different. Motility driven by ATP was rapid but restricted to the distal region of the sperm tail, whereas ADP produced slower and more fluid waves that propagated down the full flagellum. Characterization of wave patterns by tracing and superimposing the images of the flagella, quantifying the differences using digital image analysis, and computer-assisted sperm analysis revealed differences in the amplitude, periodicity, and propagation of the waves between detergent-modeled sperm treated with either ATP or ADP. Surprisingly, addition of AMP to the incubation medium containing ATP recapitulated the pattern of sperm motility seen with ADP alone. In addition to AK1 and AK2, which we previously demonstrated are present in outer dense fibers and mitochondrial sheath of the mouse sperm tail, we show that another AK, AK8, is present in a third flagellar compartment, the axoneme. These results extend the known regulators of sperm motility to include AMP, which may be operating through an AMP-activated protein kinase.

Keywords: ADP; AK8; AMP; ATP; adenine nucleotides; adenylate kinase; motility; sperm.

FIG. 1

Motility of detergent-modeled sperm reactivated by the addition of ATP or ADP. Shown are still images from video recordings at ×60 magnification (see Supplemental Movies S1 and S2) of detergent modeled sperm reactivated with solutions containing either ATP (A) or ADP (B).

FIG. 2

Tracings of individual sperm reactivated by either ATP or ADP. Sperm were reactivated with ATP (left panel) or ADP (right panel) and captured on video. At 0.06-sec intervals, the flagella were traced from individual frames of the video.

FIG. 3

Plots of angularity calculations at two different points in the flagella of detergent-modeled sperm reactivated with either ATP or ADP. Angularity was calculated by analyzing the triangle (A) formed by connecting three successive, equidistantly spaced points along the flagellum in two regions: 1) the midpiece and 2) the proximal principal piece. The points and their X and Y coordinates were identified with the program GraphClick (B). For a representative sperm reactivated with ATP or ADP, X and Y coordinates were recorded for points 1–3 (corresponding to the midpiece, as seen in green in C) and 4–6 (corresponding to the proximal region of the principal piece, depicted in red) every 0.03 sec for 5 sec. The angularities of these regions were calculated for each time point and plotted on graphs comparing the angularity of the midpiece to that of the principal piece of sperm reactivated with ATP (D) or ADP (E). Traces in green represent the midpiece region, and the traces in red correspond to the proximal region of the principal piece, plotted as a moving average over time (see Materials and Methods for detail).

FIG. 4

CASA analysis of sperm motility parameters among sperm reactivated with ATP, ADP, or ATP plus AMP. Cauda epididymal sperm were collected, extracted with Triton X-100; reactivated in the presence of millimolar amounts of ATP, ADP, or ATP plus AMP; and analyzed by CASA. Values for amplitude of lateral head displacement (ALH), curvilinear velocity (VCL), beat cross frequency (BCF, representing the rate at which the sperm head crosses the average path of the moving sperm), straight-line velocity (VSL), and linearity (LIN, derived from the ratio VSL/VCL) were recorded, averaged, and graphed with the corresponding error bars. For ALH, VCL, BCF, and VSL, there is a significant difference between the values for ATP and ADP treatment as well as between the values for ATP and ATP/AMP treatments (asterisks, P < 0.05). There is no significant difference between the values for ADP and ATP/AMP treatments for ALH, VCL, BCF, and VSL. For LIN, there is no significant difference between the values for ATP and ADP as well as ATP and ATP/AMP.

FIG. 5

Detection of AMPKα is present in both intact and detergent-modeled sperm. Protein extracts from intact mouse cauda epididymal sperm (Cd), detergent-extracted sperm (Ex), and the supernatant following detergent treatment (Sup) were analyzed by immunoblotting using a rabbit polyclonal anti-AMPKα antibody. Each band detected in Cd and Ex is the expected size for AMPKα. The numbers on the left represent molecular weights of standard proteins (×10⁻³).

FIG. 6

Ak8 mRNA expression profile. Quantitative RT-PCR (A) and RT-PCR (B) were used to determine the Ak8 expression levels in spermatogenic cells and pattern in various tissues, respectively. Gapdh expression was used as a loading control. MGC, mixed germ cells; PS, pachytene spermatocytes; RS, round spermatids; CS, condensing spermatids; Br, brain; He, heart; Ki, kidney; Li, liver; Lu, lung; Ov, ovary; Od, oviduct; SM, skeletal muscle; Sp, spleen; Te, testis; Ut, uterus.

FIG. 7

In silico analysis of the deduced AK8 protein sequence. A protein BLAST analysis as the NCBI server (http://blast.ncbi.nlm.nih.gov/Blast.cgi) identified two AK domains within the AK8 protein sequence [50]. The N-terminal (upper) and C-terminal (lower) portions were aligned to illustrate the two AK regions (brackets corresponding to amino acids 59–249 of the N-terminal portion and 270–463 of the C-terminal domain). The solid line indicates the position of the two regions that align with the canonical signature sequence for AKs. Amino acids conserved between the two AK domains are illustrated with shaded boxes. Prolines are indicated by dots (.) above the sequences; a double dot (:) indicates conserved prolines found in both AK domains. The arrows illustrate the positions of the two peptide sequences (MDATTAPHRIPPEM, amino acids 1–14, and VQVRLLQNPKDSEEYIK, amino acids 414–430) used to generate the AK8 antibody.

FIG. 8

AK8 protein expression profile. SDS-PAGE and immunoblotting of spermatogenic cells and various tissues was used to determine in which tissues AK8 was present. A) AK8 was seen in mixed germ cells, round spermatids, and condensing spermatids. B) AK8 was found in all tissues examined except liver. Immunoblots probed with anti-AK8 serum are located on the left. Immunoblots probed with preimmune serum are located on the right. MGC, mixed germ cells; PS, pachytene spermatocytes; RS, round spermatids; CS, condensing spermatids; Cd, cauda epididymal sperm; Br, brain; He, heart; Li, liver; Lu, lung; Ov, ovary; Od, oviduct; SM, skeletal muscle; Sp, spleen; Te, testis; Ut, uterus.

FIG. 9

Localization of AK8 in cauda epididymal sperm. Immunofluorescence of cauda epididymal sperm was performed to determine the localization of AK8 in sperm. AK8 was present along the full length of the sperm tail and the acrosomal region of the sperm head (top). No fluorescence was observed when sperm were probed with preimmune serum (bottom). Nomarski images are located to the right of the fluorescent images. A 10-μm bar is indicated on all images.

FIG. 10

Detergent fractionation of cauda epididymal sperm. A series of detergent extractions was performed on cauda epididymal sperm to determine if AK8 was associated with the sperm axoneme. Sperm were extracted in ice-cold PBS, 1% Triton X-100, and 1% S-EDTA. Supernatants (S) were separated from pellets (P). Samples were further processed for SDS-PAGE and immunoblotting.