Calcium regulation of the soluble adenylyl cyclase expressed in mammalian spermatozoa

Abstract

In mammals, Ca2+ and HCO3- ions play a critical role in the regulation of sperm function, most likely by regulation of cAMP levels. Mammalian germ cells contain a soluble adenylyl cyclase (sAC) with properties distinct from the well characterized membrane-bound enzymes Here we investigated whether the cyclase expressed in mature spermatozoa has the properties of sAC and whether it is regulated by Ca2+. In addition to an HCO3--dependent activation, the cyclase endogenous to human spermatozoa is stimulated 2- to 3-fold by Ca2+ in a concentration-dependent manner (EC50 approximately 400 nM). In a similar fashion, Ca2+ activates the recombinant rat and human full-length sAC with similar EC50 values. The Ca2+ stimulation was also observed when sAC was activated with HCO3-, was independent of calmodulin, and was associated with an increase in Vmax without changes in Km for ATP-Mg2+. An increase in intracellular Ca2+ by ionophore or by a muscarinic cholinergic receptor agonist increases cAMP in cells transfected with FL-hsAC, but not in mock-transfected cells. Similarly, both Ca2+ and HCO3- stimulate cAMP accumulation in human spermatozoa. These findings provide evidence that human spermatozoa express a cyclase with the properties of sAC and that Ca2+ can substitute for HCO3- in the stimulation of this enzyme, underscoring an important role for sAC in the control of sperm functions.

Fig. 1.

Detection of sAC activity in human sperm and the effect of Ca²⁺ on sAC activity. (A) AC activity in the soluble and particulate fraction of human spermatozoa was measured in the presence of 5 mM MgCl₂ with or without 25 mM . The data reported are mean ± SEM of five independent experiments with different semen samples. *, P < 0.05; **, P < 0.001. (B) Immunoprecipitation of AC activity of human spermatozoa using N terminus (N-term), C terminus (C-term), or mid-peptide (mid-pep) anti-sAC antibodies. The AC activity was measured in the immunoprecipitation pellets in the presence of 5 mM MnCl₂ as described in Materials and Methods. The data shown are mean ± SEM of four separate experiments. (C) AC activity was determined in the presence of 5 mM MgCl₂ at the indicated concentration of free Ca²⁺ in the cytosolic fractions of washed human spermatozoa. The data represent mean ± SEM of a triplicate determination of an experiment that was repeated at least five times with different human sperm samples with similar results.

Fig. 2.

Stimulation of recombinant sAC by Ca²⁺. AC activity was measured in the cytosolic fractions prepared from HEK293 cells transfected with either empty vector or FL-hsAC plasmid DNA in the presence of 5 mM MgCl₂ at the indicated concentration of free Ca²⁺ without (A) or with (B)25 mM . The data represent mean ± SEM of triplicate determination of at least four experiments performed.

Fig. 3.

An increase in intracellular Ca²⁺ stimulates cAMP accumulation in FL-hsAC-transfected HEK293 cells. HEK293 cells transfected with an empty vector or vector containing FL-hsAC-V5 were challenged for an additional 15 min with DMEM-I (basal), DMEM-I and 10 μM A23187, or DMEM-I and 10 μM carbachol. cAMP was extracted and measured by an RIA as described in Materials and Methods. *, P < 0.05 compared with basal cAMP levels. (Upper) Western blot analysis of the TCA precipitates from HEK293 cells transfected with an empty vector (mock, M) or with FL-hsAC-V5 (FL) plasmid DNA. Extracts were fractionated by electrophoresis on SDS/8% PAGE and probed with an anti-V5 antibody. A, A23187; C, carbachol. The data represent three different experiments repeated with similar results.

Fig. 4.

cAMP accumulation in sAC-expressing cells and human sperm after an increase in intracellular Ca²⁺ and . (A) HEK293 cells were transfected with empty vector or vector containing FL-hsAC-V5. Transfected cells were challenged for 15 min with an increasing concentration of carbachol in the absence or presence of 25 mM .(B) Empty vector (mock) and FL-hsAC-transfected HEK293 cells were incubated with DMEM-I with or without 25 mM and treated with 10 μM A23187. *, P < 0.05 compared with basal cAMP levels. (C) Washed human sperm (2–5 × 10⁶ cells) were incubated at 37°C in DMEM-I (basal) or DMEM-I and 25 mM with or without 10 μM A23187 for 15 min. Different letters denote a significant difference between treatments (P < 0.05). The data represent the mean ± SEM of a triplicate determination of three separate experiments.

Fig. 5.

Kinetic properties of Ca²⁺-activated sAC. Soluble extract of FL-hsAC-V5-transfected HEK293 cells were immunoprecipitated with V5 antibody, and AC activity was measured in the presence of 10 mM MgCl₂, 200 μM EGTA, and increasing concentrations of free Ca²⁺ as a function of substrate ATP-Mg²⁺. sAC kinetics were assessed at the indicated concentration of free Ca²⁺ in the absence (A) or presence (B) of 25 mM . The data represent mean ± SEM of triplicate determination of at least two experiments.