Controlling fertilization and cAMP signaling in sperm by optogenetics

Abstract

Optogenetics is a powerful technique to control cellular activity by light. The light-gated Channelrhodopsin has been widely used to study and manipulate neuronal activity in vivo, whereas optogenetic control of second messengers in vivo has not been examined in depth. In this study, we present a transgenic mouse model expressing a photoactivated adenylyl cyclase (bPAC) in sperm. In transgenic sperm, bPAC mimics the action of the endogenous soluble adenylyl cyclase (SACY) that is required for motility and fertilization: light-stimulation rapidly elevates cAMP, accelerates the flagellar beat, and, thereby, changes swimming behavior of sperm. Furthermore, bPAC replaces endogenous adenylyl cyclase activity. In mutant sperm lacking the bicarbonate-stimulated SACY activity, bPAC restored motility after light-stimulation and, thereby, enabled sperm to fertilize oocytes in vitro. We show that optogenetic control of cAMP in vivo allows to non-invasively study cAMP signaling, to control behaviors of single cells, and to restore a fundamental biological process such as fertilization.

Keywords: cAMP; calcium; capacitation; cell biology; cyclic nucleotide signaling; mouse; optogenetics; sperm.

Figure 1.. Characterization of the Prm1-bPAC mouse.

(A) Scheme of the Prm1-bPAC targeting vector. Expression of hemagglutinin (HA)-tagged bPAC is driven by the protamine 1 promoter (Prm1); arrows indicate the position of genotyping primers. (B) Genotyping by PCR. In Prm1-bPAC mice, a 213-bp fragment is amplified. The targeting vector served as a positive control (+). (C) Western blot analyzing bPAC-HA expression in testis lysates from different founder lines. Lysates from HEK cells expressing bPAC-HA served as positive control (+), wild-type testis lysates as negative control (−). (D) Western blot analyzing bPAC-HA expression in tissue lysates from male and female Prm1-bPAC mice. (E) Western blot analyzing bPAC-HA expression in testis and sperm. (F) Immunohistochemical analysis of bPAC-HA expression (left panel: transmission, right panel: fluorescence). Pictures at the bottom show a higher magnification (see white box). Sperm flagella are indicated (arrow). Cryosections of mouse testis were probed with anti-HA antibody and fluorescent secondary antibody (green), DNA was stained with DAPI (blue). Loading control for Western blots: β-tubulin. DOI: http://dx.doi.org/10.7554/eLife.05161.003

Figure 2.. Manipulation of cAMP signaling and sperm motility by light.

(A) Light stimulation of bPAC sperm for 10 min increases cAMP levels. Stimulation of SACY activity with HCO₃⁻ (1 min) evokes the same response in wild-type and bPAC sperm. (B) cAMP levels in bPAC sperm during prolonged light stimulation and after switching off the light. (C) Western blot analyzing SACY and PKA expression in wild-type and bPAC sperm. (D) Phosphorylation of PKA targets in wild-type and bPAC sperm detected with an anti-phospho-(Ser/Thr) PKA substrate antibody (p-PKA). Wild-type sperm were stimulated with 25 mM HCO₃⁻, bPAC sperm with HCO₃⁻ or light. (E) db-cAMP- (1 mM) and HCO₃⁻-induced tyrosine phosphorylation in wild-type and bPAC sperm detected with an anti-phospho-tyrosine antibody (pY). (F) Acrosome-reaction assay. Percentage of sperm that has undergone the acrosome reaction under non-capacitating conditions (no HCO₃⁻) before and after light-stimulation and under capacitating conditions (25 mM HCO₃⁻). (G) Basal flagellar beat frequency in wild-type and bPAC sperm (individual values and mean ± s.d.). (H, I) Light-induced change in flagellar beat frequency of individual bPAC sperm. (J) Average change in flagellar beat frequency after light stimulation. (K) Change in flagellar beat frequency of bPAC sperm after global or local light stimulation of the flagellum for 100 ms; see also Video 3 and 4. Data are plotted as mean ± s.d.; (n) = number of experiments, n ≥ 5 if not stated otherwise; p values calculated using Student's t test. Loading control for Western blots: β-tubulin. DOI: http://dx.doi.org/10.7554/eLife.05161.005

Figure 3.. cAMP does not evoke a Ca²⁺ influx in mouse sperm.

(A, B) Ca²⁺ signals induced by 8-Br-cAMP (10 mM; left) and db-cAMP (10 mM; right) in wild-type (A) and bPAC sperm (B) loaded with the fluorescent Ca²⁺ indicator FluoForte. Arrows indicate the addition of compounds. Light stimulation does not evoke a Ca²⁺ signal. Signals were measured in 96 multi-well plates in a fluorescence plate reader. (C) Mean signals evoked by light stimulation, cyclic nucleotide analogs, and ionomycin. (D) Illumination according to the protocol in (A, B) stimulates cAMP synthesis in bPAC sperm. Data are plotted as mean ± s.d.; (n) = number of experiments; p values calculated using Student's t test. (E, F) Ca²⁺ signals of capacitated sperm induced by cyclic nucleotide analogs (10 mM) in wild-type (E) and CatSper-null (F) sperm loaded with the fluorescent Ca²⁺ indicator Cal-520 in a stopped-flow device. Inset in (F): ionomycin control (2 µM). DOI: http://dx.doi.org/10.7554/eLife.05161.010

Figure 4.. bPAC restores fertility in mice lacking functional SACY.

(A) Nominally Ca²⁺-free medium attenuates HCO₃⁻-induced cAMP synthesis by SACY in wild-type and bPAC sperm. Inhibition of phosphodiesterases by IBMX in wild-type sperm and light stimulation in bPAC sperm increases cAMP levels without extracellular Ca²⁺. Data are plotted as mean ± s.d.; (n) = number of experiments; p values calculated using Student's t test. (B) In the absence of extracellular Ca²⁺, HCO₃⁻-induced tyrosine phosphorylation (pY) is strongly attenuated. In bPAC sperm, light stimulation is sufficient to restore tyrosine phosphorylation. (C) Light stimulation restores flagellar beating of Slc9a10-null/bPAC sperm. Flagellar waveform of Slc9a10-null/bPAC sperm before (left) and after light stimulation (right). Successive, aligned, and superimposed images creating a ‘stop-motion’ image, illustrating one flagellar beating cycle. Scale bar: 30 µm. (D) Upon light stimulation, Slc9a10-null/bPAC sperm fertilize oocytes in vitro (mean ± s.d.; (n) = total number of oocytes from three independent experiments). DOI: http://dx.doi.org/10.7554/eLife.05161.011