We recently identified a ClC-type anion channel encoded by clh- 3 that is functionally expressed in C. elegans oocytes. CLH-3 is activated during oocyte meiotic maturation suggesting that the channel plays a role in meiotic cell cycle progression, ovulation, fertilization, and/or early development. Disruption of channel expression by RNA interference has little effect on various reproductive events. However, in worms injected with
clh-3 dsRNA, we observed that ovulatory contractions of the gonadal sheath cells were initiated prematurely This suggests that CLH-3 functions in inhibitory signaling pathways that modulate sheath cell contractile activity (Rutledge et al., Curr. Biol. 11: 161-170, 2001 ). Oocytes are coupled to sheath cells by gap junctions (Hall et al., Dev. Biol. 212:101-123, 1999 ) indicating that the two cell types may communicate via electrical and chemical signals. We postulated that activation of CLH-3 during meiotic maturation depolarizes the oocyte and electrically-coupled sheath cell plasma membranes. We also postulated that depolarization modulates sheath cell contractile activity by regulating calcium influx via receptor-activated calcium channels that are triggered by depletion of IP3-sensitive intracellular calcium stores. To begin testing this model, we have developed an isolated gonad preparation. Sheath cells continue to contract for at least 1-2 h after gonad dissection. Removal of extracellular calcium completely inhibits sheath contractions. Increasing calcium influx with the ionomycin induces tonic sheath contraction. Sheath cells load readily with the fluorescent calcium probe
fluo-3, and voltage-sensitive fluorescent dyes suggesting that it may be possible to directly quantify intracellular calcium signaling events and membrane voltage during the contractile cycle. GFP reporter studies have demonstrated that the IP3 receptor gene
itr-1 is expressed in sheath cells (Baylis et al., J. Mol. Biol. 294:467-476, 1999 ). To determine whether IP3 signaling events play a role in regulating sheath cell contractions, we have begun analyzing sheath contractile activity by video microscopy in worm strains harboring mutations in
itr-1 . In wild-type worms, sheath cells exhibit a basal contraction rate of 8.6 +/- 0.7 contractions/min with a displacement of 2.3 +/- 0.01 m m. The force and frequency of sheath contractions increase dramatically 3-4 min after maturation is initiated. These ovulatory contractions reach a peak value of 21.3 +/- 1.7 contractions/min with a displacement of 4.6 +/- 0.03 m m. The JT73 nematode strain harbors a loss-of-function mutation in
itr-1 (Dal Santo et al., Cell 98:757-767, 1999). JT73 worms exhibit virtually no basal sheath contractions. Mean basal contractile activity measured in four worms over >3 h of video recording was 0.1 +/- 0.02 contractions/min. Rare basal contractions were observed, but never exceeded a rate of 1 contraction/min. The displacement of these contractions was 2.1 +/- 0.01 m m. Ovulation in JT73 worms is severely disrupted. All worms analyzed had oocytes present in the spermatheca at the beginning of video recording. The most proximal oocyte in these worms underwent meiotic maturation triggering what appeared to be ovulatory contractions. These contractions lasted 5.7 +/- 0.4 min and reached a peak value of 5.8 +/- 0.7 contractions/min with a displacement of 3.7 +/- 0.1 m m. Each worm exhibited 3-4 of these apparent ovulatory contraction cycles during the period of observation. However, no successful ovulations occurred. Ovulation was unsuccessful in the worms we analyzed because the previously ovulated oocyte failed to exit the spermatheca. Our results demonstrate that IP3 and calcium signaling pathways play critical roles in regulating sheath cell contractile activity as well as events that control the release of fertilized oocytes into the uterus.