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Proc Natl Acad Sci U S A
2021 Nov 30;11848:. doi: 10.1073/pnas.2109993118.
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Molecular mechanisms of sperm motility are conserved in an early-branching metazoan.
Speer KF
,
Allen-Waller L
,
Novikov DR
,
Barott KL
.
Abstract
Efficient and targeted sperm motility is essential for animal reproductive success. Sperm from mammals and echinoderms utilize a highly conserved signaling mechanism in which sperm motility is stimulated by pH-dependent activation of the cAMP-producing enzyme soluble adenylyl cyclase (sAC). However, the presence of this pathway in early-branching metazoans has remained unexplored. Here, we found that elevating cytoplasmic pH induced a rapid burst of cAMP signaling and triggered the onset of motility in sperm from the reef-building coral Montipora capitata in a sAC-dependent manner. Expression of sAC in the mitochondrial-rich midpiece and flagellum of coral sperm support a dual role for this molecular pH sensor in regulating mitochondrial respiration and flagellar beating and thus motility. In addition, we found that additional members of the homologous signaling pathway described in echinoderms, both upstream and downstream of sAC, are expressed in coral sperm. These include the Na+/H+ exchanger SLC9C1, protein kinase A, and the CatSper Ca2+ channel conserved even in mammalian sperm. Indeed, the onset of motility corresponded with increased protein kinase A activity. Our discovery of this pathway in an early-branching metazoan species highlights the ancient origin of the pH-sAC-cAMP signaling node in sperm physiology and suggests that it may be present in many other marine invertebrate taxa for which sperm motility mechanisms remain unexplored. These results emphasize the need to better understand the role of pH-dependent signaling in the reproductive success of marine animals, particularly as climate change stressors continue to alter the physiology of corals and other marine invertebrates.
Fig. 1. Response of sperm from the coral Montipora capitata to ammonium chloride (NH4Cl) treatment and sAC inhibition. (A) Coral sperm motility (solid line) and pHi (dashed line) after exposure of sperm in NaFSW to 20 mM NH4Cl (n ≥ 15 cells per time point). Gray shaded region indicates 99% CI of sperm initial pHi prior to activation (7.52 ± 0.05). (B) Concentration of cAMP in coral sperm in NaFSW following treatment with 20 mM NH4Cl (black squares) or control (open squares). cAMP levels are normalized to total protein. (C) Circular motility of coral sperm in NaFSW preincubated with 0.2% DMSO (black) or 50 µM sAC inhibitor KH7 (gray) before and after treatment with NH4Cl. The horizontal black bar represents the mean of n ≥ 3 replicates. (D) The median speed of coral sperm in seawater pretreated with 0.2% DMSO or 50 µM KH7. The horizontal black bar represents the mean of n = 5 replicates. Error bars in A, C, and D indicate SEM; error bars in B indicate SD; where not visible, they fall within the symbol. Significance is denoted as n.s. (no significance), *P ≤ 0.05, **P ≤ 0.01.
Fig. 2. Expression of sAC in Montipora capitata sperm. (A) Western blot of the sAC protein in sperm from three different individuals (lanes 1 to 3) shows high expression of two isoforms (sACFL, sAC110) and low expression of two isoforms (sAC55, sAC45). (Inset) High exposure of same blot. Approximate size (kDa) of each protein is indicated on the left. (B) Western blot of somatic tissues from four M. capitata adults (lanes A to D) show expression of a single sAC isoform (sAC45). (C) Diagram of the architecture of coral sperm, identified by ref. 46. The head houses the nuclei and the surrounding acrosome compartment. The flagellum contains the axoneme formed from a 9 + 2 microtubule bundle and, immediately posterior to the head, a mitochondria-rich midpiece. (D) Brightfield image of sperm. (E) Corresponding fluorescence micrograph showing localization of sAC (green), DNA (blue), and flagella (magenta; stained with anti-β-tubulin antibodies). (F) Corresponding image of coral sAC expression alone. (G) Brightfield image of a sperm cell with the flagellum extended. (H) Corresponding fluorescence micrograph highlighting coral sAC expression along the length of the flagellum. Arrowheads indicate the midpiece of the flagellum containing the mitochondrial sheath, and asterisks indicate the flagellum.
Fig. 3. A pH-dependent motility pathway is conserved in sperm from the coral Montipora capitata. (A) Diagram of the pH-sAC-cAMP motility pathway from echinoderms. Egg-derived chemoattractants bind to a guanylyl cyclase receptor (GC-A) which produces cGMP to stimulate CNGK-mediated K+ efflux and membrane hyperpolarization (Hyp). CNGK activates the H+/Na+ exchanger SLC9C1, which raises cytoplasmic pH, thereby activating sAC-dependent cAMP production and driving PKA-dependent phosphorylation of axonemal proteins controlling motility. sAC-dependent cAMP feeds back to maintain SLC9C1 activity and promotes hyperpolarization-dependent activation of HCN, which depolarizes (Dep) the cell via Na+ influx. The CatSper channel responds to both depolarization and elevated pH to generate Ca2+ influx signals that alter the flagellar waveform. (B) Expression of a predicted 40-kDa M. capitata PKA Cα catalytic subunit was confirmed by Western blot. The protein ran as a triplet, likely because of activating phosphorylations. (C) Western blot analysis also detected an increase in PKA substrate phosphorylation in sperm stimulated with 20 mM NH4Cl. Predicted domain structures of M. capitata homologs of (D) mcSLC9C1, (E) mcCatSpercomp, (F) mcGC-A, (G) mcCNGK, and (H) mcHCN. Conserved protein domains and amino acid signatures: K, kinase domain; S, transmembrane segment; NHE, sodium hydrogen exchange domain; *, includes NHE consensus sequence; VSD, voltage sensing domain; +, positively charged amino acids involved in voltage sensing; CNBD, cyclic nucleotide-binding domain; P, pore-forming domain; SF, selectivity filter; CC, coiled-coil domain; GC, guanylyl cyclase domain; KL, kinase-like domain; LB, ligand-binding domain.
Fig. 4. Evolutionary conservation of the sperm motility activation pathway across Metazoan phyla. (Left) Sperm release strategies among the phyla. Spawning refers to release of sperm into an external aquatic environment for internal or external fertilization. Copulation refers to direct sperm transfer and internal fertilization. (Middle) Phyla in which one or more species exhibit cAMP-dependent sperm motility. (Right) Phyla in which one or more species have sAC, SLC9C1, or CatSper expression confirmed in sperm (blue), encoded in the genome (teal), or absent from all available genomes (gray). White circles indicate that no data were found in the published literature.
