The mechanism underlying the ZP-induced pHi increase is unknown. To investigate the
contribution of a protein of the Na+/H+ exchanger family to the ZP pHi signaling, I studied ZP-
induced pHi signals in sperm bathed in 138 mM (control) and 0 mM extracellular Na+. Under
control conditions, ZPs evoked the characteristic pHi increase, whereas at low [Na+]o, the ZP-
evoked alkalization was abolished (ΔR/R0 = 3.7 ± 1.9 % vs. -0.2 ± 1.3 %) (Fig. 3.10a,b). The pHi
increase evoked by NH4Cl was, however, similar at high and low [Na+]o (Fig. 3.10c). These results
support the notion that the ZP-induced alkalization is mediated by Na+/H+ exchangers. A promising candidate is the sperm-specific sodium proton exchanger (sNHE) (Wang et al., 2003), which is potentially regulated by changes in VM because it contains a putative voltage-sensor
domain.
To reveal the role of sNHE in ZP signaling, I analyzed ZP-evoked pHi and Ca2+ responses in sNHE-
deficient sperm. Mixing of sNHE-/- sperm with NH4Cl evoked an alkalization, whereas ZPs did not
change pHi (Fig. 3.11a). This finding supports the notion that the sNHE mediates the ZP-induced
alkalization. Moreover, in sNHE-/- sperm, also the ZP-evoked Ca2+ response (1.1 ± 2.5 %), but not the Ca2+ response evoked by K8.6 or ionomycin, was abolished (Fig. 3.11b,c). Taken together,
Fig. 3.10: ZP-evoked pHi responses at high and low extracellular Na+
(a,b) Change in pHi evoked by 0.5 ZP/µl (dark green) or 10 mM NH4Cl (light green) in capacitated
sperm bathed in (a) 138 mM or (b) 0 mM Na+ buffer. Mean ± 95 % CI (dashed traces), n = 3. (c) Mean signal amplitudes; error bars indicate + SD (n = 4).
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these experiments suggest that alkalization mediated by the sNHE is crucial for the ZP-evoked Ca2+ response.
However, sNHE forms a signaling complex with SACY, and deletion of the Slc9c1 gene also disrupts SACY expression. Therefore, sNHE-/- sperm not only lack the sNHE, but also SACY function. To disentangle whether the lack of ZP signaling in sNHE-/- sperm is due to a lack of
Fig. 3.11:ZP-induced pHi and Ca2+i response in sNHE-/- sperm
(a) Change in pHi evoked by 0.5 ZP/µl (dark green) or 10 mM NH4Cl (light green) in capacitated sNHE-/-
sperm. Mean ± 95 % CI (dashed traces), n = 4. (b) Change in [Ca2+]i evoked by 0.5 ZP/µl in capacitated
sNHE-/- mouse. Mean ± 95 % CI (dashed traces), n = 4. (c) Change in [Ca2+]i evoked by K8.6 (light blue)
or 2 µM ionomycin (grey) in capacitated sNHE-/- sperm. Mean ± 95 % CI (dashed traces), n = 7.
Fig. 3.12: Light-induced activation of bPAC stimulates cAMP synthesis in bPACtg/+sNHE-/-
sperm
Left panel: In wildtype sperm, SACY converts ATP into the second messenger cAMP. Right penal:
SACY is lost in sNHE-/- sperm. To rescue cAMP synthesis, a light-activateable adenylate cyclase from bacteria (bPAC) is crossed into sNHE-/- sperm.
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sNHE, SACY, or a combination of both, I used the following approach: It has been reported that incubation of sNHE-/- sperm with membrane-permeable cAMP derivates restores the loss of SACY, and, thereby, sperm motility (Wang et al., 2007). Therefore, I tested whether restoring cAMP levels in sNHE-/- sperm rescues the ZP responses. We have generated transgenic mice expressing a bacterial, photoactivatable adenylate cyclase (bPAC) exclusively in sperm (Jansen et al., 2015). Stimulating of bPAC-expressing sperm with blue light increased cAMP levels (Jansen et al., 2015). We crossed these mice with sNHE-/- mice to generate bPACtg/+sNHE-/- mice. First, we tested whether cAMP production in sNHE-/- sperm, and thereby sperm motility, can be rescued by light- induced stimulation of cAMP synthesis (Fig. 3.12). To this end, the motility of sNHE-/- sperm was studied via dark-field microscopy. Fig. 3.13a shows the waveform of the flagellar beat before and after activation of bPAC (experiment was performed by Vera Jansen). In the
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Fig. 3.13: Infertility of sNHE-/- mice can be rescued by an artificial increase of intracellular
cAMP levels
(a) Light stimulation restores flagellar beating of sNHE-/-bPACtg/+ sperm. Flagellar waveform of sNHE-/- bPACtg/+ sperm before (left) and after light stimulation (right). Color-coded frames are superimposed creating a ‘stop-motion’ image, illustrating one flagellar beating cycle; scale bar: 30 µm. (b) Acrosome reaction evoked by 1 ZP/µl or 2 µM ionomycin in capacitated wildtype and sNHE-/- sperm before and after preincubation with 5 mM db-cAMP. Data are shown as mean + SD percentage of acrosome- reacted (AR) sperm normalized to the buffer-treated control, n = 4. (c) Non-fertilized and fertilized oocyte in one- and two-cell state, respectively. (d) Rate of two-cell state oocytes after incubation of oocytes with sperm from wildtype, sNHE-/-, and bPACtg/+sNHE-/- sperm. The bPACtg/+sNHE-/- sperm were illuminated for 90 min with blue light prior to the in vitro fertilization experiment (mean + SD, n = total number of oocytes from three independent experiments).
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dark, bPACtg/+sNHE-/- sperm were immotile. However, after activating bPAC by light, sperm regained a symmetric flagellar beat, resulting in a regular, progressive movement. These results
confirm that the immotility of sNHE-/- sperm is due to the lack of cAMP synthesis via SACY. Next, I studied acrosome reaction in sNHE-/- sperm in the absence and presence of db-cAMP
(Fig. 3.13b). In the absence of cAMP, both ZPs and ionomycin failed to evoke the acrosome reaction in sNHE-/- sperm. Similar results have been reported for SACY-/- sperm (Xie et al., 2006). Bathing sNHE-/- sperm in db-cAMP restored the ZP- and ionomycin-evoked acrosome reaction, demonstrating that SACY-dependent cAMP synthesis, but no the sNHE function, is required for the acrosome reaction.
Moreover, I tested whether light-induced cAMP synthesis rescues the infertility phenotype of sNHE-/- sperm. In an in vitro fertilization assay, capacitated wildtype or bPACtg/+sNHE-/- sperm were incubated with wildtype oocytes. After 24 hours, the number of two-cell-stage oocytes was quantified as a measure for fertilization (Fig. 3.13c). Wildtype sperm fertilized about 30 % of
Fig. 3.14: Increase in cAMP rescues ZP-induced pHi and [Ca2+]i increase in sNHE-/- sperm
(a) Change in pHi evoked by 0.5 ZP/µl (dark green) or 10 mM NH4Cl (light green) in capacitated sNHE-/-
sperm after preincubation with 5 mM db-cAMP. Mean ± 95 % CI (dashed traces), n = 4. (b) Mean signal amplitudes; error bars indicate + SD(n = 4). (c) Change in pHi evoked by 0.5 ZP/µl (dark green)
or 10 mM NH4Cl (light green) in bPACtg/+sNHE-/- sperm after light stimulation. (d) Change in [Ca2+]i
evoked by 0.5 ZP/µl in capacitated sNHE-/- sperm after preincubation with 5 mM db-cAMP. Mean ± 95 % CI (dashed traces), n = 4. (e) Mean signal amplitudes normalized to the maximal response evoked by 2 µM ionomycin; error bars indicate + SD(n = 4). (f) Change in [Ca2+]i evoked by 0.5 ZP/µl
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the oocytes, whereas in bPACtg/+sNHE-/- sperm the fertilization rate was 2 % (Fig. 3.13c). This residual fertilization rate reflected the spontaneous first mitotic division that occurs in some oocytes due to in vitro maturation (Bałakier & Casper, 1991; Cheng et al., 2012). However, after light stimulation, bPACtg/+sNHE-/- sperm were able to fertilize the oocyte, resulting in a fertilization rate of 16 % (Fig. 3.13d). These results demonstrate that light-induced cAMP synthesis restores the fertility of sNHE-/- sperm.
Finally, I studied ZP-induced pHi and Ca2+ responses in sNHE-/- sperm that were bathed in db-
cAMP and in bPACtg/+sNHE-/- sperm stimulated with blue light to activate bPAC (Fig. 3.14). Both, db-cAMP and light-stimulated cAMP synthesis via bPAC restored the pHi and Ca2+ response in
sNHE-/- sperm, demonstrating that the sNHE does not underlie the ZP-induced alkalization- signaling in mouse sperm.