The "soluble" adenylyl cyclase in sperm mediates multiple signaling events required for fertilization

Abstract

Mammalian fertilization is dependent upon a series of bicarbonate-induced, cAMP-dependent processes sperm undergo as they "capacitate," i.e., acquire the ability to fertilize eggs. Male mice lacking the bicarbonate- and calcium-responsive soluble adenylyl cyclase (sAC), the predominant source of cAMP in male germ cells, are infertile, as the sperm are immotile. Membrane-permeable cAMP analogs are reported to rescue the motility defect, but we now show that these "rescued" null sperm were not hyperactive, displayed flagellar angulation, and remained unable to fertilize eggs in vitro. These deficits uncover a requirement for sAC during spermatogenesis and/or epididymal maturation and reveal limitations inherent in studying sAC function using knockout mice. To circumvent this restriction, we identified a specific sAC inhibitor that allowed temporal control over sAC activity. This inhibitor revealed that capacitation is defined by separable events: induction of protein tyrosine phosphorylation and motility are sAC dependent while acrosomal exocytosis is not dependent on sAC.

Figure 1. Soluble AC Is in Male Germ Cells and Sperm

(A) AC assays of germ cells and sperm. The amount of cAMP produced in the presence of magnesium (Mg), magnesium + bicarbonate (Mg+B), and magnesium + forskolin (Mg+FSK) was assayed using soluble (S) and particulate (P) proteins prepared from pachytene spermatocytes, round spermatids, condensing spermatids, and sperm. (B) Immunoblot (using biotinylated sAC monoclonal antibody R21) of immunoprecipitates (using sAC monoclonal antibody R5 recognizing a nonoverlapping epitope) from wild-type (+/+) and sAC null (−/−) testis cytosol. The first lane is a protein extract from sAC_t-overexpressing cells (sAC_t is the M_r ~50,000 isoform generated by alternative splicing). (C) Immunofluorescent analysis of sAC in noncapacitated wild-type sperm using monoclonal antibody R21 (Zippin et al., 2003). Arrows refer to midplece region. (D) Corresponding phase image of (C). (E) Immunofluorescent analysis of sAC in capacitated wild-type sperm using the R21 antibody. Arrow refers to annular region. (F) Corresponding phase image of (E).

Figure 2. Sperm from sAC Null Animals Have Defects in Motility

(A) Motility analysis of sAC null sperm. Sperm from +/+ and −/− animals were collected and capacitated in the absence (solid bars) and presence (open bars) of 1 mM dbcAMP. Progressive motility was determined as described at 1.5 hr. n = 4 for both +/+ and −/− animals. *p < 0.05. (B) Flagellar angulation of sAC null sperm. (a) A wild-type sperm. (b) A sAC null sperm in the process of forming a hairpin at the annulus. (c) A sAC null sperm in which the head has bent back 180° onto the tail. In all three photomicrographs, the head (arrowhead) and annulus (arrow) are denoted. (C) Schematic diagram of a sperm showing the annulus, a region that demarcates the midpiece from the principal piece and where the flagellar angulation occurs. Diagonal lines in the midpiece represent the mitochondrial sheath.

Figure 3. Sperm from sAC Null Animals Show an Aberrant Protein Tyrosine Phosphorylation Pattern

Sperm from +/+ and −/− animals were collected. Both noncapacitated (NC) and capacitated (CAP) sperm were prepared in the presence and absence of 1 mM dbcAMP. Proteins were solubilized, separated by gel electrophoresis, and assayed for the presence of protein tyrosine phosphorylation by immunoblotting with anti-phosphotyrosine antibody. (A) Protein from +/+ sperm. The band at M_r ~116,000 (arrow) is hexokinase that is constitutively tyrosine-phosphorylated (Kalab et al., 1994). (B) Protein from −/− sperm. Both noncapacitated and capacitated sperm from −/− animals had an aberrant pattern of protein tyrosine phosphorylation pattern that did not change with the addition of dbcAMP.

Figure 4. KH7-Treated Sperm Show a Reduction in cAMP Levels

Noncapacitated (NC) and capacitated (CAP) sperm were prepared and either left untreated or treated with 10 μM or 50 μM KH7. The amount of cAMP produced was determined, and data points correspond to averages of duplicate determinations with standard deviations indicated. This experiment was repeated two times with similar results.

Figure 5. Effect of KH7 on Protein Tyrosine Phosphorylation in Sperm

Epididymal sperm were collected and incubated under noncapacitating (NC) and capacitation (CAP) conditions in the presence and absence of KH7. Proteins were solubilized, separated by gel electrophoresis, and assayed for the presence of protein tyrosine phosphorylation by immunoblotting with the anti-phosphotyrosine antibody. (A) Sperm coincubated with various concentrations of KH7. The band at M_r ~116,000 (arrow) is hexokinase, which is constitutively tyrosine phosphorylated. (B) In the presence of 10 μM KH7 and 1 mM dbcAMP, the prototypical protein tyrosine phosphorylation pattern was observed.