DEV024521 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Immunofluorescent staining for NOS of primary cultures (endothelial cells) derived from explants of human oviduct (ampullary region). (A,C,E,G) Phase images. (B,D,F,H) Corresponding immunofluorescence images. Cultures were approximately 24 hours old and retained beating cilia. Constitutive (eNOS, nNOS) and inducible (iNOS) isoforms were clearly present in all cells. No staining was detectable in control incubations in which the primary antibody was omitted. Fluorescence pictures were all taken with the same settings. Scale bars: 50 µm.

• Supplemental Figure S2 -

  Fig. S2. Immunofluorescent staining for NOS of COV434 (human granulosa) cells. (A,C,E,G) Phase images. (B,D,F,H) Corresponding immunofluorescence images. Constitutive NOS isoforms (eNOS, nNOS) were clearly present in all cells but inducible NOS (iNOS), though detectable, appeared to be present at lower levels. No staining was detectable in control incubations in which the primary antibody was omitted. Fluorescence pictures were all taken with the same settings. Scale bars: 50 µm.

• Supplemental Figure S3 -

  Fig. S3. (A) Human cumulus stained with DAF (green fluorescence), demonstrating NO synthesis by all or nearly all cells. (B) Phase image of mouse oocyte-cumulus, overlaid with fluorescent image from DAF staining, showing production of NOS by a proportion (∼10%) of cells. (C) Phase image of oviductal explant (ampullary region), overlaid with fluorescent image from DAF staining. NO production is occurring throughout the explant. Arrows show beating cilia that are visible where they extend beyond the edge of the explant.

• Supplemental Figure S4 -

  Fig. S4. Dose-dependence of the action of NO on Ca²⁺_i in human spermatozoa. Traces show the responses of seven separate cells to sequential addition of 1 µM, 10 µM and 100 µM spermine NONOate (shown by bars above the traces). Only one cell (yellow trace) responds to 1 µM NONOate. At 10 µM, many cells show a slow increase in Ca²⁺_i and at 100 µM the response is increased and occurs in many cells as irregular transients or oscillations. In these experiments, fewer than 20% of cells showed no response (blue trace).

• Supplemental Figure S5 -

  Fig. S5. Simple model to illustrate the relationship between increase in fluorescence induced by NO Δ fluorescence (NONOate) and fall in fluorescence caused by subsequent application of DTT Δ fluorescence (DTT). (A) The saturating relationship of fluorescence to Ca²⁺, modelled using the equation y=1-(1/exp(x) (where x represents Ca²⁺ and y represents fluorescence). (B,C) Two alternatives for the relationship between the rise in Ca²⁺ caused by NO (ΔCa²⁺ NONOate) and the fall in Ca²⁺ caused by DTT (ΔCa²⁺ DTT). In B, the amplitude of the response to DTT is determined by the preceding action of NONOate (85% reversal of NONOate effect±randomly generated ‘noise’). In C, the effect of DTT is to reduce Ca²⁺_i by a constant increment (±randomly generated ‘noise’). (D,E) Changes in fluorescence that would be recorded (based upon the relationship shown in A) in scenarios B and C, respectively. The results in Fig 5C (and four other experiments) resemble those in D.

• Movie 1 -

  Movie 1. Stimulation of flagellar activity by progesterone in the presence of NO•. Movie shows four cells imaged in the presence of 100 µM spermine NONOate (added ∼8 minutes previously). Cells were imaged at 1 Hz and total length of recording is 5.1 minutes. Yellow marker (top left) shows point at which progesterone (3 µM) was added (∼1 minute into recording). After progesterone application, the excursion of the flagellum increases, resulting in noticeable movement of the head in some of the cells. This increase in flagellar activity is prolonged (see Fig. 6D), consistent with the prolonged/oscillatory effect of progesterone on Ca²⁺_i under these conditions (Fig. 6A,B).

• Movie 2 -

  Movie 2. Stimulation of flagellar activity by progesterone without pre-treatment with NO. Movie shows five cells, imaged at 1 Hz and total length of recording is 8.33 minutes. Yellow marker (top left) shows point at which progesterone (3 µM) was added. After progesterone application, the excursion of the flagellum increases in some transiently in some cells, but the amplitude and (more particularly) the duration of this effect are brief compared with that seen in the presence of 100 µM NONOate.