Espinal-Enríquez J et al. (2017), Network model predicts that CatSper is the main...

ECB-ART-45595

Sci Rep 2017 Jun 26;71:4236. doi: 10.1038/s41598-017-03857-9.

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Network model predicts that CatSper is the main Ca2+ channel in the regulation of sea urchin sperm motility.

Espinal-Enríquez J , Priego-Espinosa DA , Darszon A , Beltrán C , Martínez-Mekler G .

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Spermatozoa sea urchin swimming behaviour is regulated by small peptides from the egg outer envelope. Speract, such a peptide, after binding to its receptor in Strongylocentrotus purpuratus sperm flagella, triggers a signaling pathway that culminates with a train of intracellular calcium oscillations, correlated with changes in sperm swimming pattern. This pathway has been widely studied but not fully characterized. Recent work on Arbacia punctulata sea urchin spermatozoa has documented the presence of the Ca2+ CatSper channel in their flagella and its involvement in chemotaxis. However, if other calcium channels participate in chemotaxis remains unclear. Here, based on an experimentally-backed logical network model, we conclude that CatSper is fundamental in the S. purpuratus speract-activated sea urchin sperm signaling cascade, although other Ca2+ channels could still be relevant. We also present for the first time experimental corroboration of its active presence in S. purpuratus sperm flagella. We argue, prompted by in silico knock-out calculations, that CatSper is the main generator of calcium oscillations in the signaling pathway and that other calcium channels, if present, have a complementary role. The approach adopted here allows us to unveil processes, which are hard to detect exclusively by experimental procedures.

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Genes referenced: LOC100887844 LOC100893907 LOC576642 LOC582000

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	Figure 1. Model-II signaling Pathway and Logical Network of the Speract-Activated Signaling Pathway (SASP). (A) Scheme of the signaling pathway triggered by speract in S. purpuratus sperm flagellum. The SASP starts with the binding of speract to its receptor and after several steps, which include changes in membrane potential, oscillations in [Ca 2+]i are attained. (B) Biochemical events related to (A). (C) Network model of the signaling pathway. Each node on the network represents an element of the pathway. Black arrows indicate activation; red lines indicate inhibition and the dashed yellow arrows can both activate and inhibit depending on the value of voltage V. Voltage can have a hyperpolarized state, represented with a value of 0; resting potential, 1 and a depolarized state, which is represented with a value of 2.
	Figure 2. Time series of the network dynamics of the [Ca 2+]i under the blockage of the different Ca 2+ channels present in the sea urchin sperm flagellum: HVA-LVA and CatSper. The [Ca 2+]i curve with all nodes present (WT) is depicted in black. Red line corresponds to the [Ca 2+]i dynamics after blockage of the CatSper channel. The blue line represents the [Ca 2+]i nodes of the network dynamics without both HVA and LVA channels. The X axis units are number of iterations of the network dynamics. The Y axis units are arbitrary, indicative of relative [Ca 2+]i node values.
	Figure 3. Network representation of the signaling pathway with CatSper channel as the only source of intracellular Ca 2+ concentration. Notice the absence of the LVA and HVA channels previously depicted in Fig. 1. Also note the new direct link between pH, voltage and Ca 2+ node with the CatSper channel, highlighted with bold arrows. This network constitutes Model-III.
	Figure 4. [Ca 2+]i dynamics of the network model with and without the PDE node. Panel A shows a typical experimental measurement of [Ca 2+]i florescence after exposure to speract. The control curve is depicted in black; the red curve represents the calcium level with speract + IBMX, a known PDE blocker. Note the considerable decrease in the Ca 2+ induced by PDE blockage. Panels B,C and D are the time series of the Ca 2+ level for the control curve (black) and under deletion of the PDE node (red) for Models I, II and III respectively. In the time series of panel B, the average calcium level is slightly lower and its oscillations smaller under PDE deletion. Model-II (panel C) remarkably displays only small changes. However, for Model-III (panel D), the calcium level under PDE-blockage is drastically reduced.
	Figure 5. [Ca 2+]i dynamics of the network deleting the pH node for the three models. In panel A for Model-I calcium dynamics, with all nodes present (black) and without the pH node (red), notice that there is hardly any difference in the level of calcium between the two situations. A similar situation is present in panel B for Model-II. The case of Model-III is shown in panel C, the calcium fluctuations completely disappear in the absence of pH, this is so, because the pH directly controls the CatSper dynamics.
	Figure 6. [Ca 2+]i dynamics of the network model with and without the CaKC channel for Models II and III. In panel A for Model-II the [Ca 2+]i node dynamics with all nodes present is shown in black and without the CaKC channel in red. Notice the higher calcium concentration in the red curve. A longer period for the WT is manifest in panel B by the appearance of an additional lower frequency Fourier mode. For panels (C) and (D), the Model-III calcium dynamics shows a decrease in the calcium level (as observed in the experiments with Iberiotoxin)16 as well as a more elaborate temporal behaviour in the Fourier power spectrum with the appearance of a longer period component not present in the WT case (also observed in the mentioned experiment).
	Figure 7. Comparison between experiments on the effect of niflumic acid (NFA) and the network dynamics of Model-III. (A) Experimental [Ca 2+]i curves using 100 nM of speract (black) and 100 nM of speract + 10 μM of NFA (red). Notice that the average [Ca 2+]i mean, amplitude, maximum peak and interval between peaks is higher in the red curve (with NFA) than in the WT curve. (B) Ca 2+ time series generated from Model-III with all nodes present in black, and in red the “NFA case”, with HCN, CaCC channels blocked and CaKC, CatSper channels over activated. The green curve is placed as a guide to the eye for an envelope of both experiment and model series. (C) Fourier spectra of the above time series, with same colour code. Notice the more elaborate temporal behaviour and higher period component in the red curve.
	Figure 8. Catsper participates in the speract signaling cascade. Fluo-4 labelled S. purpuratus sea urchin sperm diluted (1:10) in 1CaSW pH 7.0, were further diluted (5 μl) in 800 μl ASW pH 7.8. After recording the fluorescence for 10 seconds, 1 nM speract (A and C), 10 μM Mibefradil (Mibe) (A), or 5 μM NNC-055-396 (NNC) (C), were added. Channel blockers were incubated for 1 min. In (A and C) arrows indicate speract additions. (B and D) Summary of experiments performed as in (A) or (C) respectively, assessing the fluorescence change in the maximum after additions. The Fluo-4 normalized fluorescence (%) was obtained considering the fluorescence obtained after adding 0.05% Triton-X100 as 100%. Bars represent the mean ± s.e.m. (B) n = 3–8. (D) n = 4–5. p < 0.001 and *p < 0.02.

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Accili, Inhibition of the hyperpolarization-activated current (if) of rabbit SA node myocytes by niflumic acid. 1996, Pubmed