MPK-1/ERK regulatory network controls the number of sperm by regulating timing of sperm-oocyte switch in C. elegans germline

Abstract

The precise regulation of germline sexual fate is crucial for animal fertility. In C. elegans, the production of either type of gamete, sperm or oocyte, becomes mutually exclusive beyond the larval stage. Hermaphrodites initially produce sperm and then switch to produce oocytes. This change of fate during germline development is tightly controlled by several regulators. In C. elegans hermaphrodites, FBF-1 and FBF-2 (>95% identical, members of the Pumilio RNA-binding protein family) proteins function redundantly to promote the sperm-oocyte switch. Here, we demonstrate that loss of LIP-1 (dual specificity phosphatase) in fbf-1(ok91) single mutants leads to excess sperm production due to a delayed sperm-oocyte switch. This phenotype was dramatically rescued by depletion of MPK-1 (an ERK homolog). In contrast, loss of LIP-1 in fbf-2(q738) single mutants leads to a premature sperm-oocyte switch and loss of sperm. Notably, fbf-1 fbf-2; lip-1 triple mutants produce excess sperm. These results suggest that the MPK-1/ERK regulatory network, including FBF-1, FBF-2, and LIP-1, controls the number of sperm by regulating the timing of the sperm-oocyte switch in C. elegans.

Keywords: Caenorhabditis elegans; FBF RNA-binding proteins; LIP-1; MPK-1/ERK; Sperm number; Sperm-oocyte switch.

Fig. 1

C. elegans sexual fate decision and its regulators (A–C) Schematic germline phenotypes caused by aberrant sperm/oocyte switch. (A) Wild-type hermaphrodite, (B) Fog (for feminization of the germ line), (C) Mog (for masculinization of germline). In each cartoon, anterior is left, dorsal is up. The gonad consists of two arms that share a central uterus. Sperm (dark blue); Oocytes (light pink). (D) Simplified version of the hermaphrodite germline sex determination pathway. Low and high refer to levels of above gene activities.

Fig. 2

Germline phenotypes of fbf-1(ok91); lip-1(zh15) and fbf-2(q738); lip-1(zh15) mutants.(A) Germline phenotypes of adult hermaphrodites. Sp+Oo, making both sperm and oocytes; ExSp, excess sperm production (>500 sperm per gonadal arm); LeSp, less sperm production (<100 sperm per gonadal arm). Germline phenotype was scored 2 days after L4. (B–E) DIC pictures of adult worms (1 day after L4). Solid grey lines, boundary of gonad; broken lines, sperm; sold black circles, embryos; asterisks, oocyte nuclei. Scale bars: 10 um. (G) Sperm number in hermaphrodite worms. Statistical analysis was performed using ANOVA. n, number of gonadal arms scored. p<0.0001 (***). No statistically significant difference (ns).

Fig. 3

MPK-1 activity in the distal germline is required for continued sperm production. (A) The inhibition of MPK-1 by FBF-1 and LIP-1 at post-transcriptional and post-translational levels [22]. (B and C) Immunohistochemistry using anti-RME2 (a marker for oocytes) and anti-SP56 (a marker for sperm) antibodies. Scale bars: 10 um. (D) Germline phenotypes of adult worms (1 day after L4). (E–H) Immunohistochemistry using anti-MAPK(YT) antibody that recognizes C. elegans activated MPK-1. Broken red lines indicate a region where activated MPK-1 proteins were detected. Asterisks (*), distal end. Scale bars: 10 um.

Fig. 4

MPK-1 acts upstream of FOG-1 and FOG-3. (A) Germline phenotype of adult worms. n*, number of dissected gonad arms scored. (B) Immunohistochemistry using anti-RME2, anti-SP56, and DAPI. Scale bars: 10 um. (C–F) Proposed working models.