RHGF-1 regulates the activity of RHO-1 and does not affect calcium dynamics. (A–D) Based on GCaMP3 movies, the number of Ca²⁺ peaks, the number of Ca²⁺ peaks per second, and the amount of time after oocyte entry required to reach half of the maximum or maximum Ca²⁺ signal was determined for control and rhgf-1(ok880) Ca²⁺ traces. N ≥ 12. Error bars are ± SEM. (A, B, and D) Statistical comparisons were performed by two-tailed unpaired t test with Welch’s correction. (C) Statistical comparison was performed by two-tailed unpaired t test. (E) Representative kymographs of control and rhgf-1(ok880) movies are shown with time of entry, distal neck closure, and time the sp-ut valve opens and closes indicated. Kymographs were generated by averaging over the columns of each movie frame (see Materials and methods). Scale bars: vertical scale bar, 1 sec; horizontal scale bar, 10 µm. Time elapsed is indicated on the y-axis. Full movies are in Video 2. (F) Live imaging of spermatheca-specific RHO-1 biosensor (AHPH::GFP) during embryo transit through spermatheca in control and rhgf-1(RNAi) worms. Arrowheads point to the distal end of the spermatheca, where RHO-1 activity is high in the control and low in the rhgf-1(RNAi). Dashed line marks the area where RHO-1 biosensor was quantified. Scale bar, 20 µm. Full movies are in Video 3. (G) Quantification of RHO-1 biosensor fluorescence intensity during ovulation and embryo transit in control and rhgf-1(RNAi) worms. The x-axis (time) was normalized according to distinct events so that all traces begin at the time when the distal valve closes behind the ovulating oocyte (“Oocyte entry complete”) are aligned at the time the sp-ut starts to open (“Embryo exit initiation”) and aligned again when the sp-ut valve closes behind the exiting embryo (“Embryo exit complete”). N = 6. (H) Maximum intensity projections of spermathecae expressing GFP tagged to ACT-1 (actin) in control, rho-1(RNAi), and rhgf-1(RNAi) worms. Arrows point to wavy actin. Scale bar, 10 µm. (I) Tortuosity of actin bundles in the spermathecae of control, rho-1(RNAi), and rhgf-1(RNAi) worms. Tortuosity is defined as the actual length between two ends of an actin bundle divided by the shortest distance between them. N ≥ 12. Error bars are ± SEM. Statistical comparisons were performed by One-way ANOVA-Tukey’s multiple comparisons test. Stars designate statistical significance (*** P < 0.001, ** P < 0.01, ns P > 0.05). N, number of samples analyzed.