CRISP1 as a novel CatSper regulator that modulates sperm motility and orientation during fertilization

Abstract

Ca(2+)-dependent mechanisms are critical for successful completion of fertilization. Here, we demonstrate that CRISP1, a sperm protein involved in mammalian fertilization, is also present in the female gamete and capable of modulating key sperm Ca(2+) channels. Specifically, we show that CRISP1 is expressed by the cumulus cells that surround the egg and that fertilization of cumulus-oocyte complexes from CRISP1 knockout females is impaired because of a failure of sperm to penetrate the cumulus. We provide evidence that CRISP1 stimulates sperm orientation by modulating sperm hyperactivation, a vigorous motility required for penetration of the egg vestments. Moreover, patch clamping of sperm revealed that CRISP1 has the ability to regulate CatSper, the principal sperm Ca(2+) channel involved in hyperactivation and essential for fertility. Given the critical role of Ca(2+) for sperm motility, we propose a novel CRISP1-mediated fine-tuning mechanism to regulate sperm hyperactivation and orientation for successful penetration of the cumulus during fertilization.

Figure 1.

CRISP1 expression in the female tract. (A) Total RNA from ovary (ova), oviduct (ovi), uterus (ut), cumulus cells (cu), and eggs (egg) of Crisp1^+/− (HT) or Crisp1^−/− (KO) mice was subjected to RT-PCR using specific primers for CRISP1. In both cases epididymis (ep) was used as control. Products were separated on 2% agarose gels and stained with ethidium bromide for visualization. (B) Protein extracts (100 µg) obtained from ovary, oviduct, and uterus (left) and from cumulus cells and eggs (right) as well as from the epididymis (0.2 µg; used as control) from Crisp1^+/− or Crisp1^−/− mice were subjected to SDS-PAGE and Western blotting using anti-CRISP1 (top) or anti-tubulin (bottom) as primary antibodies. (C) COC from Crisp1^+/− and Crisp1^−/− animals were washed, fixed, and subjected to indirect immunofluorescence using anti-CRISP1 as primary antibody (green). DNA staining with propidium iodide is shown in red. Bar, 30 µm.

Figure 2.

Participation of cumulus CRISP1 in fertilization. (A) COC from Crisp^+/− (HT) and Crisp1^−/− (KO) mice were coincubated with capacitated Crisp1^+/− or Crisp1^−/− sperm for 3 h and then stained with Hoechst 33342 for evaluation of fertilization. Results represent the mean ± SEM of three independent experiments. *, P < 0.05 vs. Crisp1^+/− COC; **, P < 0.001 vs. all groups. (B) Crisp1^+/− and Crisp1^−/− COC were coincubated for 15 min with capacitated Crisp1^+/− or Crisp1^−/− sperm previously loaded with Hoechst 33342, and the number of sperm observed within the cumulus (top) was determined (bottom). Results represent the mean ± SEM of five independent experiments. *, P < 0.05 vs. Crisp1^+/− COC. The total numbers of eggs analyzed in each case are indicated in parentheses. Bar, 50 µm.

Figure 3.

Sperm orientation and motility in the presence of CRISP1. (A) Capacitated sperm were placed in one well of a modified Zigmond chamber and CRISP1 (1 pM to 10 µM), DTT-treated and heat-denatured CRISP1 (1 µM; DTT/Φ), or recombinant CRISP1 (10 µM; rec) were loaded in the second well. Medium alone was used as negative control and both progesterone (100 pM; P) and cumulus-conditioned medium (CM) were used as positive controls. After 15 min, the percentage of oriented sperm toward the corresponding gradients was calculated by analyzing sperm trajectories. In all cases, results represent the mean ± SEM of at least three independent experiments in which >150 sperm trajectories per experiment were analyzed. *, P < 0.05; **, P < 0.005 vs. medium. (B) Percentages of hyperactivated (left), transitional (middle), or linear (right) patterns of motility for sperm exposed to either CRISP1 (1 µM) or medium (control; top) and for oriented and nonoriented cells within the CRISP1-exposed group (bottom). In all cases, results represent the mean ± SEM of seven independent experiments in which at least 100 sperm trajectories per experiment were analyzed. **, P < 0.005; *, P < 0.05.

Figure 4.

CRISP1 inhibits the macroscopic cationic currents in testicular sperm. (A) Representative whole-cell patch clamp currents recorded on a testicular sperm at the cytoplasmic droplet. The currents were evoked applying voltage steps (20 mV) from a holding potential of 0 mV to test potentials ranging from −100 to +100 mV in cationic solution (HS media with impermeable anions). The protocol used for eliciting cationic currents in A and D is shown below traces in D. Representative whole-cell currents under control conditions (left) and after adding 10 µM CRISP1 using a picospritzer close to the sperm (right). (B) Mean I-V curves from experiments as in A. Results represent the mean ± SEM of four independent experiments. (C) Mean I-V relationship of cationic currents in the presence of 10 µM of heat-denatured CRISP1 (CRISP1Φ) compared with the control. The currents were elicited with the same voltage protocol as in A. (D) Currents recorded from sperm in the Cs⁺ recording solution. Representative whole-cell currents under control conditions (left) and after adding 10 µM CRISP1 (right). (E) I-V curves show inhibition by 10 µM CRISP1 of the control sperm Cs⁺ currents. (F) Control Cs⁺ whole-cell currents recorded with the same solutions as in D applying the voltage protocol in the inset (left) were stimulated by 300 µM menthol (middle). The stimulated current was inhibited by 10 µM CRISP1 (right). (G) Mean I-V curves from experiments as in F. Results represent the mean ± SEM of three experiments; in some cases, the SEM bars were smaller than symbols.

Figure 5.

CRISP1 inhibits CatSper channels in cauda epididymal sperm. (A) CRISP1 inhibition of CatSper was evaluated measuring E_m (see Materials and methods) in Whitten’s media after adding 3.5 mM EGTA, which depolarizes noncapacitated sperm caused by Na⁺ influx partly through CatSper. ΔE_m represents the difference between E_m after EGTA addition (E_mD) and before (resting E_m [E_mR]) for control sperm (n = 13) or sperm incubated for 10 min with saline solution either alone (vehicle, n = 4) or containing 10 µM CRISP1 (n = 11), heat-denatured CRISP1 (CRISP1Φ; n = 6), 1 µM NNC 55-0396 (n = 7), 500 µM Ni²⁺ (n = 4), or 10 µM HC-056456 (n = 3). Results are expressed as the mean ± SEM of the number of indicated independent experiments. *, P < 0.05; **, P < 0.001 vs. control. (B) Whole-cell currents elicited by a voltage ramp from a holding potential of 0 mV (see protocol in top panel) in physiological solution (HS) or in Cs⁺ divalent-free (Cs⁺ DVF) medium either alone or containing CRISP1 10 µM. Note the strong inhibition effect at both negative and positive potentials (top). Under the same experimental conditions, heat denatured CRISP1 (10 µM; CRISP1Φ) had no effect along the tested voltage range (bottom). (C) CRISP1 (10 µM) inhibited ∼50% of the CatSper current at both negative and positive voltages (−80 and +80 mV) and heat-denatured CRISP1 had no effect at any voltage. Results represent mean ± SEM of five different sperm.

Figure 6.

Schematic model for the behavior of sperm exposed to a CRISP1 gradient. When sperm swim along increasing CRISP1 concentrations, there is an inhibition of Ca²⁺ channels (i.e., CatSper/TRPM8) and a consequent lower entry of Ca²⁺ into the cells. This leads to lower sperm hyperactivation and higher linearity levels that maintain sperm oriented toward the positive gradient. If sperm no longer swim in increasing CRISP1 concentrations, there is no inhibition of Ca²⁺ channels and, thus, a higher Ca²⁺ influx. This results in higher hyperactivation levels that help sperm to find the positive attractant gradient again. The dotted lines indicate the dynamics of the process.