MPK-1 ERK signaling is necessary for pachytene progression and oocyte meiotic maturation (Church et al., 1994; Lee et al., 13th International C. elegans Meeting, 2001). Strong loss-of-function/null mutations in
lin-45 RAF,
mek-2 MAPKK and
mpk-1 ERK result in hermaphrodites that fail to produce either sperm or oocytes and instead germline nuclei are arrested in pachytene. In the distal meiotic prophase germline of wild-type animals, germ cells (nucleus with surrounding plasma membranes except on the side facing the central cytoplasmic core) are packed in a hexagonal pattern on the surface of the gonadal tube. In
lin-45,
mek-2 and
mpk-1 mutants, the hexagonal germ cell packing is disrupted and instead pachytene arrested nuclei and surrounding plasma membranes are found in clumps, often in the center of the gonadal tube. We call this a Pac phenotype, for pachytene arrest and clumped nuclei and membranes. A similar Pac phenotype is observed in mutant males. Under certain partial loss-of-function conditions, where pachytene progression and oocyte differentiation is normal, we observed hermaphrodites with feminized germlines (proximal germ cells developing as oocytes instead of sperm). The partial loss-of-function conditions were: a) viable escapers from
lin-45 or
mpk-1 RNAi; b)
mek-2(
n1859) (Kornfeld et al., 1995); and c) trans-heterozygotes of
mpk-1 null and
mpk-1(ts) (Lackner and Kim, 1998) at the permissive temperature. These results raise the possibility that the Pac phenotype observed in males and in the proximal germline of hermaphrodites for
lin-45,
mek-2 and
mpk-1 strong loss-of-function/null mutants is the result of the combined effect of a sexual fate transformation followed by pachytene arrest during oogenesis. In situ hybridization using a probe to
rme-2 mRNA, which encodes the oogenesis specific yolk receptor, was used to test this possibility. In wild-type L4 hermaphrodites, germ cells in the proximal gonad, most of which are in pachytene, are undergoing spermatogenesis and lack
rme-2 mRNA while germ cells in the proximal third of the distal gonad contain
rme-2 mRNA. By contrast, germ cells throughout the proximal gonad and into the distal gonad have
rme-2 mRNA in L4
mpk-1 null hermaphrodites. In
mpk-1 null males,
rme-2 mRNA is found throughout the region showing the Pac phenotype, unlike in wild-type males. Thus,
mpk-1 is required for the male germ cell fate in both hermaphrodites and males. Additional experiments indicate that
mpk-1 is functioning in the germline, not in the soma, for male germline sex determination and that
mpk-1 activity is required continuously. Epistasis experiments indicate that
mpk-1 is acting downstream or in parallel to
tra-2 in the sex determination pathway, as
tra-2 null;
mpk-1 null double mutant germlines are feminized.
mpk-1 may not be required for male somatic sex determination as sexual transformation in the adult soma (yolk synthesis) has not been observed. Accumulation of MPK-1 and the appearance of activated MPK-1 is sexually dimorphic. An affinity purified C-terminal fraction of the SC94 (Santa Cruz) antisera is specific for germline MPK-1 as germ cells do not stain in the null mutant. MPK-1 is found at high levels throughout the germline of wild-type adult hermaphrodites. By contrast, in wild-type males, MPK-1 is found at significantly lower levels and only in the distal most germ cells, up to about the distal third of the pachytene region. ERK MAP kinases are activated by phosphorylation on a specific Thr and Tyr motif, which can be detected by the MAPKYT mAb (Sigma; Miller et al., 2000). In hermaphrodites, activated MPK-1 is found at high levels in the 2 to 3 most proximal oocytes and at moderate levels in the proximal half of pachytene. In males, activated MPK-1 is only found at very low levels in the transition zone and the first few pachytene germ cells, a region where activated MPK-1 is not observed in adult hermaphrodites. Thus, MPK-1 is activated sex specifically and to different extents in three spatially distinct regions of the C. elegans germline.