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. 2007 May 29;104(22):9325-30.
doi: 10.1073/pnas.0611296104. Epub 2007 May 17.

A sperm-specific Na+/H+ exchanger (sNHE) is critical for expression and in vivo bicarbonate regulation of the soluble adenylyl cyclase (sAC)

Dan Wang  1 , Jie Hu, I Alexandru Bobulescu, Timothy A Quill, Paul McLeroy, Orson W Moe, David L Garbers
Affiliations

A sperm-specific Na+/H+ exchanger (sNHE) is critical for expression and in vivo bicarbonate regulation of the soluble adenylyl cyclase (sAC)

Dan Wang et al. Proc Natl Acad Sci U S A. .

Abstract

We previously identified a sperm-specific Na(+)/H(+) exchanger (sNHE) principally localized to the flagellum. Disruption of the sNHE gene in mice resulted in absolute male infertility associated with a complete loss of sperm motility. Here, we show that the sNHE-null spermatozoa fail to develop the cAMP-dependent protein tyrosine phosphorylation that coincides with the functional maturation occurring upon incubation in capacitating conditions in vitro. Both the sperm motility defect and the lack of induced protein tyrosine phosphorylation are rescued by the addition of cell-permeable cAMP analogs, suggesting that cAMP metabolism is impaired in spermatozoa lacking sNHE. Our analyses of the bicarbonate-dependent soluble adenylyl cyclase (sAC) signaling pathway in sNHE-null sperm cells reveal that sNHE is required for the expression of full-length sAC, and that it is important for the bicarbonate stimulation of sAC activity in spermatozoa. Furthermore, both codependent expression and coimmunoprecipitation experiments indicate that sNHE and sAC associate with each other. Thus, these two proteins appear to be components of a signaling complex at the sperm flagellar plasma membrane. We propose that the formation of this complex efficiently modulates intracellular pH and bicarbonate levels through the rapid and effective control of sAC and sNHE activities to facilitate sperm motility regulation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
sNHE-null spermatozoa have reduced basal cAMP levels that are insensitive to bicarbonate stimulation. (A) Sperm cells from wild-type (Wt) and sNHE-null mice were collected in a bicarbonate-free medium. cAMP content was measured by using RIA. Data represent the mean ± SD (n = 4). (B) Wild-type and sNHE-null spermatozoa were diluted in a bicarbonate-containing medium and incubated at 37°C. At each indicated time point, an aliquot of sperm was collected and analyzed for cAMP content. Data represent the mean ± SD (n = 3).
Fig. 2.
Fig. 2.
Bicarbonate-stimulated soluble adenylyl cyclase (AC) activity is reduced in sNHE-null spermatozoa. Whole-sperm-cell lysates from wild-type or sNHE-null mice were used to estimate in vitro adenylyl cyclase activity in the presence of 40 mM NaCl or 40 mM NaHCO3. Data represent mean ± SD (n = 4). (Inset) Fold activation by bicarbonate in each experiment was calculated. Data represent mean ± SD (n = 4).
Fig. 3.
Fig. 3.
The sNHE-null spermatozoa contain no detectable full-length sAC protein. (A) Northern blot of total RNA (10 μg each) from wild-type, heterozygous, and sNHE-null testes with a mouse sAC cDNA probe (Upper) or a cyclophilin cDNA probe (Lower). (B) Immunoblot of total sperm cell lysate (2 × 106 cells) from wild-type or sNHE-null mice with R21 monoclonal antibody (200 ng/ml). (C) Full-length sAC transcript exists in sNHE-null testes. (Upper) Location of the sense primer (framed) relative to the sAC full-length cDNA. The gray letters show 29 of the 56 nucleotides that are missing in truncated sAC cDNA. (Lower) Relative sACfl mRNA levels in wild-type (+/+) and sNHE-null (−/−) testes determined by real-time PCR. Expression of sACfl mRNA was normalized to 18S rRNA and is reported relative to that of wild-type testis. Data represent mean ± SD (n = 3). (D) The same blot in B was stripped and reprobed with anti-human sAC polyclonal antibody (1:5,000).
Fig. 4.
Fig. 4.
Reciprocal facilitation of expression of sNHE and sAC in HEK293F cells. (A) Coexpression of the V5-tagged sACt1 enhanced expression of the myc-tagged full-length sNHE (sNHE) and the chimeric sNHE [N(1–3)s] in HEK293F cells as determined by immunoblot with anti-myc (1 μg/ml) (Top), anti-V5 (200 ng/ml) (Middle), and anti-tubulin (100 ng/ml) (Bottom) antibodies. (B) Wild-type and chimeric sNHE enhance V5-tagged sACfl protein expression in HEK293F cells. (Upper) Total cell lysates (80 μg per sample) were used for immunoblotting with anti-V5 antibody. (Lower) The same blot was stripped and reprobed with anti-tubulin antibody. (C) sNHE enhances V5-tagged sACt1 protein expression in HEK293F cells. (Upper) Total cell lysates (80 μg per sample) were used for immunoblotting with anti-V5 antibody. (Lower) Total cell lysates (50 μg per sample) were used for immunoblotting with anti-tubulin antibody. (B and C) The relative intensity of each sAC band was quantified with NIH ImageJ software.
Fig. 5.
Fig. 5.
sNHE and sAC coimmunoprecipitate from coexpressing cultured cells and spermatozoa. (A) Full-length chimeric sNHE protein associates with sAC proteins. (Left) myc-tagged chimeric sNHE was expressed in HEK293F cells coexpressing empty vector, V5-tagged sACfl, or V5-tagged sACt1. Total cell lysates were subject to immunoprecipitation (IP) with anti-V5 antibody, and the captured proteins were analyzed by immunoblot (IB) with anti-Myc antibody. (Right) V5-tagged sACfl was expressed in HEK293F cells coexpressing empty vector or myc-tagged chimeric sNHE. Total cell lysates were subject to immunoprecipitation with anti-myc antibody, and the captured proteins were analyzed by immunoblot with anti-V5 antibody. (B) C-terminal tail of sNHE interacts with sACt1 protein. Myc-tagged C-terminal tails of sNHE were expressed in HEK293F cells with or without V5-tagged sACt1. (Upper) Total cell lysates were immunoprecipitated with anti-V5 antibody, and the precipitated proteins were analyzed by immunoblot with anti-myc antibody. (Lower) Crude total cell lysates were analyzed by immunoblot with anti-myc antibody. (C) sNHE interacts with sAC in sperm cells. Sperm cell lysate was immunoprecipitated with anti-sNHE polyclonal IgG (lane 1) or preimmune IgG (lane 2). The precipitated proteins were analyzed by immunoblot with anti-human sAC polyclonal antibody.
Fig. 6.
Fig. 6.
sNHE is a NHE. (A) Detection of sNHE mRNA in stable cell lines. NHE-null cells were stably transfected with empty vector, myc-tagged sNHE, or myc-tagged chimeric sNHE constructs. sNHE mRNA in these cells was determined by real-time PCR and compared with that of wild-type mouse testis. The stable cells were acid-loaded repetitively, and the result is shown below the graph. (B) The stable cells expressing the highest sNHE message displayed NHE activity. Representative tracings of sodium-dependent pH recovery after an acid load in NHE1-expressing fibroblasts and in NHE-null fibroblasts stably transfected with either chimeric sNHE construct or empty vector. The experiment was repeated three times with three to four replicates per condition.
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