C. elegans oocytes express an inwardly rectifying Cl - channel that is activated by cell swelling and is encoded by the ClC gene
clh-3 . The biophysical characteristics of CLH-3 are virtually indistinguishable from heterologously expressed mammalian ClC-2. CLH-3 and ClC-2 share 40% amino acid identity. We have suggested that CLH-3 and ClC-2 are orthologs. The amount of swelling required to activate CLH-3 varies by >50-60 fold between different oocytes. CLH-3 in small, early stage oocytes requires substantially more volume increase to activate compared to larger, later stage oocytes. Upon completion of growth, oocytes undergo meiotic maturation and are ovulated and fertilized. In full-grown, maturing oocytes, CLH-3 is constitutively activated. Mean whole-cell Cl - currents at -70 mV were -3.2 +/- 0.3 pA/pF and -14.6 +/- 2.9 pA/pF in non-maturing and full-grown, maturing oocytes, respectively. These findings suggest that either completion of growth or induction of maturation activate the channel. To determine the role of cell growth versus meiotic maturation in CLH-3 activation, we carried out patch clamp studies on oocytes isolated from
fog-2 mutant worms. Mutations in certain C. elegans sex determination genes such as
fog-2 block sperm production in hermaphrodites ( Schedl and Kimble, Genetics 119:43-61, 1988 ). The presence of sperm is required for normal progression of meiotic maturation. Late-stage
fog-2 oocytes reach full-grown size, but arrest in diakinesis for many hours to days ( McCarter et al., Dev Biol 205:111-128, 1999 ). Since ovulation is triggered by maturation, full-grown oocytes accumulate in the gonad of
fog-2 worms. We reasoned that if completion of growth activates CLH-3, then the channel should be active in late-stage
fog-2 oocytes. However,
fog-2 oocytes exhibited no channel activity. Meiotic maturation was induced in
fog-2 oocytes by injecting recombinant Major Sperm Protein (MSP)38 into the uterus. CLH-3 was constitutively activated in late stage, maturing oocytes from MSP38-injected
fog-2 worms (Cl - current = -39.7 +/- 1.5 pA/pF). These results indicate that CLH-3 is activated by induction of meiotic maturation. To further examine the role of maturation in channel activation, we carried out a series of experiments on wild type oocytes undergoing maturation in vitro . Shortly before maturation begins, the nucleus increases in size and migrates to the cell periphery. Meiotic maturation is marked by Nuclear Envelope Breakdown (NEBD). Oocytes with centrally located nuclei were never observed to undergo maturation after isolation from the gonad. However, when oocytes with off-center nuclei were isolated and placed in a bath chamber, all of them underwent maturation within 2-10 min (mean time to beginning of NEBD = 6.2 +/- 1 min; n=9). Cell volume did not change significantly prior to and during the maturation process. In separate experiments, we patch clamped oocytes with off-center nuclei and oocytes that had undergone NEBD in vitro . CLH-3 activity was not detected in oocytes with intact nuclear envelopes ( Cl - current = -2.1 +/- 0.3 pA/pF ). However, in oocytes undergoing maturation in vitro , CLH-3 activity was detected immediately upon obtaining whole-cell access. After membrane rupture, CLH-3 continued to activate until the current reached a stable plateau level ( Cl - current = -44.4 +/- 6.2 pA/pF ). Taken together, our results demonstrate clearly that CLH-3 is activated during meiotic maturation rather than after completion of oocyte growth. We are currently characterizing the signaling pathways responsible for channel activation.