Description

Vitellogenins are the most conserved family of yolk proteins in oviparous animals, wherein the proteins are typically synthesized in extraovarian organs, secreted to fluid of a circulatory system and taken up by the ovary (Ramos et al. 2022). In C. elegans, vitellogenins are synthesized in the intestine, transported into the body cavity, and taken up by developing oocytes through receptor-mediated endocytosis to provide a source of nutrients for the developing embryo (Grant and Hirsh 1999; Hall et al. 1999; Kimble and Sharrock 1983). C. elegans has six vitellogenin genes (vit-1 – vit-6), which combine to make up four major yolk proteins: YP170A and YP170B, YP115, and YP88 (Perez and Lehner 2019). To visualize the process of yolk uptake by oocytes, many published studies have made use of worms expressing an integrated transgene coding for a fusion protein of green fluorescent protein (GFP) with VIT-2, the most abundantly expressed C. elegans vitellogenin (Grant and Hirsh 1999). This fusion protein construct has been used in several studies as not only a means of visualizing yolk uptake via receptor-mediated endocytosis, but also as a means of quantifying embryo content and brood size in a variety of genetic backgrounds (Balklava et al. 2016; Grant and Hirsh 1999; Van Rompay et al. 2015). Here we report that wild-type strains expressing this VIT-2::GFP construct have a phenotype of reduced embryo production, resulting in fewer embryos contained in adult animals at any time as well as a markedly reduced brood size compared to N2 animals. We urge future studies to consider the effects of the vit-2::gfp transgene on normal reproduction when making conclusions about data generated using these strains, and to compare all experimental findings to a wild-type strain where possible.

Upon using pwIs23 [vit-2::gfp] expressing worms (RT130) in our own experiments, a discrepancy in the size of young adult VIT-2::GFP worms compared to wild-type N2 worms was noted, as well as a difference in the number of worms on each plate during routine passaging. To more accurately assess whether these differences were due to variations in strain backgrounds, RT130 was back-crossed in our laboratory, and wild-type worms expressing the VIT-2::GFP transgene were obtained. Upon inspection of these individual worms, the VIT-2::GFP worms appeared to contain fewer embryos in utero than wild-type worms (Figure 1A). To quantify the apparent differences in embryo content, an egg counting assay was performed where synchronized day one adult worms were dissolved individually in 20% bleach solution. Upon dissolution of the cuticle, the remaining embryos from each worm were counted. For RT130 (Grant and Hirsh 1999) as well as our two independently crossed VIT-2::GFP strains (HAH20 and HAH21), the embryo count per worm was about half to one-third that of wild-type N2 worms (Figure 1B).

To determine whether the observed differences in worm population during passaging were the result of a decreased brood size, the total brood size of wild-type and VIT-2::GFP (HAH20) worms over their reproductive adulthood was determined. Two synchronized L4 worms were plated on each of 4 plates per strain and moved to fresh plates each day. Offspring on each plate were counted for two consecutive days after removal of the adult worms. Mirroring the embryo content, VIT-2::GFP worms produced about half as many offspring during their lifetimes as wild-type worms (Figure 1C). As egg-laying for both strains started, peaked, and finished on the same days (Figure 1D), it is unlikely that the differences in embryo content/brood size result from either a difference in egg-laying behavior or a developmental delay, but rather reflect an overall reduction in embryo production throughout the lifetime of VIT-2::GFP worms.

While the mechanism behind this reduction in brood size is still unclear, it is possible that the unusual uptake of VIT-2::GFP by coelomocytes may be related (Paupard et al. 2001). Looking back at the literature, one study compared the reproductive output of vit-2::gfp worms to wild-type animals (Seah et al. 2016). The supplemental data in that study indicates the brood size of RT130 was approximately 50 worms less than a wild-type strain, but the assay did not reach statistical significance. Our analysis of several strain backgrounds and quantification of 50-100 worms in multiple assays revealed a significant brood size defect, which we feel is important to keep in mind for those using VIT-2::GFP strains in future studies. We also feel that conclusions from past studies that did not verify results from VIT-2::GFP strains in wild-type worms or include a non-GFP control should be cited with caution or verified accordingly.