Description

Most cellular processes require the coordinate activities of multiple gene products. These collaborations vary with different contexts. mel-28 encodes a conserved multi-functional nucleoporin required for nuclear envelope integrity, the post-mitotic rebuilding of the nuclear pore, and the proper segregation of chromosomes during mitosis (Galy et al. 2006; Fernandez and Piano 2006). Loss of mel-28 results in strict maternal-effect embryonic lethality. Thus homozygous mutant embryos produced from heterozygous mothers show typical wild-type development until they become adults and produce normal-sized broods of inviable embryos. This suggests that mel-28 is required for embryonic development but is dispensable for post-embryonic development and adult processes such as fertility. To identify genes that function collaboratively with mel-28, we did an RNAi screen to find genes that cause fertility defects in mel-28 mutant animals but not wild-type animals (Fernandez et al. 2014). Among the genes identified were those encoding components of the minus-end-directed microtubule motor dynein.

Cytoplasmic dynein is the major minus-end directed microtubule motor in eukaryotes. It consists of six subunits, each present within the complex as dimers (Pfister et al. 2006). The dynein heavy chain, encoded by dhc-1 in C. elegans, is the largest subunit of the dynein complex. The head region of this subunit binds microtubules directly and is required for ATP hydrolysis. The tail domain binds to the remainder of the dynein subunits that in turn serve as adapters that permit interactions with cargoes. Dynein motor activity is required for diverse functions including transport of membrane-bound vesicles, centrosome separation, mitotic chromosome segregation, nuclear movement, virus transport, and organelle trafficking (Pfister et al. 2006).

To study the defects caused by simultaneous disruption of MEL-28 and dynein, we generated double mutants (Fernandez et al. 2014). We used a temperature-sensitive allele of dhc-1(or283) (Hamill et al. 2002; O’Rourke et al. 2007) in which dynein function is disabled at 26 °C and functions normally at 16 °C. dhc-1; mel-28 double mutants shifted to the restrictive temperature as L4s produce a small brood size compared to each single mutant and compared to the wild-type (Fernandez et al. 2014).

In wild-type C. elegans, diakinesis-stage oocytes undergo meiotic maturation in response to maturation-promoting factor (MPF), which is in turn positively regulated by the phosphatase CDC-25.2 and negatively regulated by the Myt1 kinase WEE-1.3 (Burrows et al. 2006; Kim et al. 2010; Huelgas-Morales and Greenstein, 2018). Since defects in oocyte maturation cause fertility defects, we sought to determine whether the reduced brood size we observed in dhc-1; mel-28 double mutants is coincident with oocyte maturity defects. To accomplish this, we used immunolocalizations to study oocyte maturity markers in dhc-1; mel-28 mutants, dhc-1 mutants, mel-28 mutants, and wild-type animals.

In the proximal gonad of wild-type hermaphrodites, oocytes are aligned in a linear assembly-line fashion such that the -1 oocyte, which is the oocyte closest to the spermatheca, is the most mature and the next to be ovulated and fertilized (McCarter et al. 1999; Huelgas-Morales and Greenstein 2018). Oocytes more distal to the spermatheca are progressively less mature. As oocytes mature their nucleoli are dismantled and their chromatin accumulates phosphorylated histone H3 (Burrows et al. 2006). Thus, in wild-type animals, the -1 oocyte typically lacks the nucleolus and its chromatin has phosphorylated histone H3.

To determine whether the reduced fertility in dhc-1; mel-28 double mutants is associated with oocyte maturity defects, we studied the proximal gonad using oocyte maturity markers. We used DAO-5 antibodies, which detect the Nopp140 component of the nucleolus (Korcekova et al. 2012; Hadwiger et al. 2010) as a marker for oocyte immaturity, and antibodies against phosphorylated histone H3 (Hendzel et al. 1997) as a marker for mature oocytes. In wild-type animals, dhc-1 single mutants, and mel-28 single mutants, the -1 oocyte rarely retained the nucleolar marker DAO-5 but nearly always had chromatin with phosphorylated histone H3. However, in dhc-1; mel-28 double mutants, the -1 oocyte frequently retained the DAO-5 immaturity marker and failed to accumulate the phosphorylated histone H3 maturity marker (Figure 1A-D). This shows that in dhc-1; mel-28 double mutants, oocytes occupying the most proximal positions do not always achieve mature status, suggesting that dynein and MEL-28 contribute in parallel to the maturity program in the oogenic gonad.

About 25% of the dhc-1; mel-28 gonads had a disorganized proximal region with several oocytes clustered near the spermatheca instead of being aligned one-by-one (Figure 1A). In some cases two oocytes essentially occupied the same position; For example, in the dhc-1; mel-28 gonad in Figure 1A, the -2 and -3 oocyte cannot be discerned. In all cases when the proximal gonads showed this disorganization, the oocytes occupying equivalent positions displayed the same combination of markers, making it irrelevant which oocyte was interpreted to be in which position. All gonads that showed this proximal clustering of oocytes also exhibited -1 oocyte maturity failure. In addition we observed some dhc-1; mel-28 animals with immature proximal oocytes and no obvious morphological disorganization.

While reduced fertility in dhc-1; mel-28 double mutants is a consistently penetrant phenotype (Fernandez et al. 2014), we observed the aberrant immature status of the -1 oocyte in only about 40% of the dhc-1; mel-28 gonads we studied (Figure 1B and 1C). This suggests that oocyte maturity defects are not the primary cause of the low fertility phenotype. We favor the idea that the oocyte maturity defects described here are a secondary effect of a problem caused by the simultaneous compromise of mel-28 and minus-end directed motor activity.