Cold shock induces a terminal investment reproductive response in C. elegans

Acute cold shock causes drastic phenotypic alterations

The duration of cold exposure for young adult hermaphrodite C. elegans at 2 °C is negatively correlated to post-shock survival rates¹⁵. Wild-type hermaphrodite worms exposed to a 4-h cold shock (CS) do not initially display high mortality rates (Fig. 1a); this allows observation of a range of phenotypic transitions as they recover from the limited-duration cold stress at their preferred temperature of 20℃. One of the most striking phenotypes exhibited in post-cold shock (post-CS) animals during the recovery period is a dramatic decrease in pigmentation in the normally highly pigmented intestine, so that the body becomes almost entirely clear (Fig. 1b, c)¹⁵. This is often accompanied by motor and reproductive disruptions such as mobility loss, withering of the gonad arms, decreased number of internal embryos, and the eventual death of about 30% of the population (Fig. 1a–d)¹⁵. It should be noted that these phenotypic responses do not appear to be due to any relative heat shock following the transition from 2 to 20 °C as the expression of GFP-tagged HSP-4 (heat shock protein) is not induced following cold shock (Fig. 1e). Neither is the reduced pigmentation following cold shock due to a period of starvation presumably experienced by the worms while they are at 2 °C. At this extreme cold temperature, the worms enter a “chill coma” in which pharyngeal pumping and virtually all other movement ceases^15,16; however, a total absence of food for a similar time period does not induce a comparable clearing phenotype (Supplemental Fig. S1). Interestingly, some CS wild-type animals regain pigmentation after clearing; these worms do not die and display a general reversal of the other negative impacts of cold shock (Fig. 1b)¹⁵. We sought to better understand the factors regulating the post-CS recovery program in wild-type worms, focusing particularly on the functional role of pigmentation loss and the genetic components involved in producing it.

Cold shock induces lipid reallocation from somatic tissues to the germline

Since pigmentation in the C. elegans intestine is due in part to the presence of lipid-storing fat droplets¹⁷, we wondered whether the decrease in pigmentation in cold-shocked worms corresponds to a depletion of intestinal lipid supplies, potentially as a result of increased metabolism meant to fuel post-cold shock recovery. Using Nile Red, which accumulates and fluoresces in hydrophobic environments¹⁸, as an indicator of lipid content, we therefore analyzed the fat stores of worms 12 h following CS (note that all cold shocks were performed for 4 h at 2℃). We indeed observed a significant decrease (P < 0.0001) in the average lipid content per worm (Fig. 2a); visually, this presents as an overall reduction in average fluorescence that is most striking in the intestine (Fig. 2c). However, we also unexpectedly noted that the lipid content of the gonad appears to concomitantly increase, marked by the presence of fluorescent (and therefore lipid-rich) internal embryos in the gonads of many cold-shocked worms that were mostly absent in their non-cold shocked counterparts (Fig. 2b, c). These embryos are somewhat sporadic, usually accounting only for a proportion of all embryos in the germline and are interspersed with non-fluorescent embryos. Importantly, the percentage of fluorescent internal embryos per worm was found to be elevated two-fold in CS worms relative to that of nonCS controls (Fig. 2b). These observations suggest that rather than just metabolically depleting lipid supplies, C. elegans may also reallocate lipids from the intestine to the germline. To further analyze this process spatiotemporally, we performed a time course of Nile Red staining following cold shock (Fig. 2c). After exit from cold shock and the corresponding chill coma^15,16, most worms gradually resume movement over the first 30 min of recovery at 20℃. At this point, the distribution of Nile Red-staining lipids is indistinguishable from non-cold shocked controls, indicating that lipid reallocation occurs after, rather than during, the cold shock. By 4 h post-cold shock, we observed visually decreased but rarely absent fluorescence in the intestine; at this time, many worms have brightly fluorescing proximal oocytes, suggesting that lipids are beginning to relocate from the intestine into the germline. This process of reallocation appears to continue over the next 14 + hours, with many 12 h-recovered worms containing fluorescent embryos and oocytes but only slight intestinal fluorescence, and most 18 h-recovered worms fluorescing almost exclusively in the embryos. Though it is unclear what other factors (e.g. metabolism) may contribute to the overall pigmentation loss, these observations suggest that the dramatic decrease in intestinal pigmentation results in large part from the reallocation of lipids from somatic tissues to the germline during the recovery phase following a severe cold shock.

Thermosensation is required to initiate the cold shock response

We next asked how wild-type C. elegans perceive and interpret cold shock as a signal for lipid reallocation. The cGMP-gated channel subunits TAX-2 and TAX-4 are important for thermosensation and acquired cold tolerance in worms, acting specifically in the ASJ neurons^11,19,20. We reasoned that worms might also rely on a neuronal mechanism of sensation that acts through the TAX-2/TAX-4 channel to induce the cold shock response in the absence of prior cold exposure. tax-2(p671); tax-4(p678) double loss-of-function mutants were therefore tested for sensitivity to cold shock. Not only do these mutants show 100% survival following cold shock (Fig. 3a), but, unlike wild-type worms, they also do not display any significant difference (P > 0.9999) in overall lipid content 12 h after CS, nor the striking lipid reallocation phenotype characterized by large numbers of fluorescent embryos (Fig. 3b–d). Interestingly, despite this resilience to shock, tax-2(p671); tax-4(p678) mutants still show decreased internal embryo quantities, though not to the same degree as N2 worms (Fig. 3e), suggesting that cold exposure has some additional impact on fertility independent of this thermosensation pathway. Taken together, though, these data support the hypothesis that fat reallocation as a stress response following CS may depend on a neuronal mechanism for induction. Since TAX-2/TAX-4 plays a role in temperature sensation, we speculate that canonical cold thermosensation is required for lipid reallocation to take place.

SKN-1 promotes cold stress resistance

After determining a requirement for neuronal signaling in cold-shock-induced lipid reallocation, we next wondered what intermediate factors were needed to translate the cold stimulus into a signal to mobilize resources. The master regulatory transcription factor SKN-1/Nrf2 coordinates a return to homeostasis following a variety of stresses, including oxidative stress⁴, pathogen infection^21,22, and osmotic stress²³. We therefore predicted that skn-1 would perform a similar function during cold stress recovery. Indeed, skn-1(lax188) gain-of-function mutants²⁴ are highly resistant to cold shock, with nearly 100% survival rates 96 h post-CS (Fig. 4a). Consistent with a retention of overall lipid stores by Nile Red Staining (Fig. 4b,d), these mutants did not contain significantly greater numbers of fluorescing internal embryos following cold shock (P > 0.9999), though they did have a marginal reduction in the number of internal embryos (Fig. 4d–f). Conversely, skn-1(mg570) loss-of-function mutants displayed a wild-type cold shock response, with significant reallocation (P < 0.00001) of lipids to the germline and loss of somatic fats (Fig. 4c–f). Because mg570 eliminates only the skn-1a mRNA isoform²⁵, but skn-1(lax188gf) activates both skn-1a and skn-1c, we additionally tested the CS response of skn-1(zj15) loss-of-function mutants, which are reported to have a 76% reduction in both skn-1a and skn-1c mRNA levels²⁶. We observed a similar clearing phenotype (Supplemental Fig. S2); however, these animals appeared slow-growing and less robust, which may have contributed to more substantial CS-induced lethality. Taken together, these data suggest that the skn-1a and perhaps also the skn-1c isoforms are not required for lipid mobilization in response to cold shock. However, SKN-1a/c activation protects cold-shocked worms from recovery phase lethality and prevents lipid reallocation.

Lipid reallocation results from upregulated vitellogenesis following cold shock

The process governing the normal movement of lipids from the soma to the germline is vitellogenesis, requiring the vitellogenin proteins coded for by vit-1–6 genes. Once coupled to somatic lipid supplies, the resulting lipoprotein complexes shuttle to the germline²⁷. We hypothesized that the lipid mobilization induced by cold shock is a result of the normal vitellogenesis machinery being commandeered as part of a stress response.

One of the major vitellogenins is VIT-2, which generates a 170 kD yolk protein product known as YP170B²⁸. We predicted that loss of vit-2 would reduce lipid reallocation following CS. Since preventing reallocation in our previous mutants also increased post-CS survival, we expected that vit-2 loss of function would enhance protection from CS. Consistent with these hypotheses, impairment of vitellogenesis in vit-2(ok3211) loss-of-function mutants produced phenotypes similar to tax-2(p671); tax-4(p678) animals. In addition to maintaining high survival rates, CS vit-2(ok3211) mutants did not undergo a substantial loss of intestinal lipid supplies, nor did they exhibit a marked increase in internal embryo fluorescence (despite a modest decrease in internal embryo counts) (Fig. 5a–e).

Since other vitellogenin transcripts produce different lipoprotein forms for movement to the germline, we decided to additionally test whether a member of the YP170A-generating class of vitellogenins would recapitulate our results with vit-2. To this end, we analyzed the CS phenotypes of vit-5(ok3239) loss-of-function animals. Indeed, we found that as in vit-2 mutants, survival was rescued in these animals following CS, corresponding with a retention of somatic lipids and inhibition of embryonic lipid reallocation (Fig. 6a–c,e). As with other mutants that prevent lipid reallocation, there was still an effect of cold in reducing overall embryo output 12 h post-CS, hinting that temperature impacts fertility independent of the other reproductive alterations exhibited by wild-type worms (Fig. 6d).

Cold shock response is a form of a terminal investment

Our data thus far suggest that upon recovery from acute CS, wild-type C. elegans induce a reproductive phenotype whereby somatic lipid supplies are massively relocalized to the germline using the normal vitellogenesis machinery. This appears to come at the expense of the parent’s own mortality, as retaining somatic lipids by preventing thermosensation or vitellogenesis is sufficient to rescue survival. Such a trade-off between survival and reproduction is redolent of the terminal investment hypothesis (reviewed in Gulyas and Powell, 2019²), which predicts that in some instances of acute stress where the likelihood of death is high, organisms can preferentially funnel resources to reproduction to maximize reproductive fitness at the cost of survival.

To determine whether cold stress-associated phenotypes in C. elegans are an example of such a process, we eliminated all potential for reproductive investment by assaying sterile glp-1(e2141) and glp-1(q231) worms to ask whether these worms still exhibited lipid loss or death upon cold shock. Strikingly, the absence of a germline in these animals completely prevented both intestinal clearing and death, again confirming that lipid movement from the soma to the germline is associated with parental lethality (Fig. 7a). If resource reallocation to the progeny is indeed meant to increase reproductive fitness in inclement conditions, there should be some benefit to the progeny of CS worms that offspring of nonCS parents do not receive. We speculated that in the case of environmental CS, a sudden, seasonal, cold to warm cycle might signal likelihood of future cold conditions that would impede the ability of embryos to hatch and survive to reproductive age. We therefore devised an assay to test for the relative fitness of embryos in cold conditions depending on whether they came from nonCS or CS parents and were thus more likely to have received lipid provisioning. To do this, embryos were collected from nonCS and CS parents within a 2 h time window when post-CS lipid reallocation seems to peak and subsequently exposed them to a cold stress of 24 h. We then assessed hatching rates 24 h following the embryonic cold shock. Excitingly, embryos coming from parents that had experienced cold shock prior to reproduction exhibited a small but significant increase in hatching rates relative to their counterparts from nonCS hermaphrodite parents. Furthermore, preventing lipid reallocation by impairment of vitellogenesis in either vit-2(lf) or vit-5(lf) was able to substantially ablate this effect, suggesting that the improved survival is attributable specifically to lipid investment in the F1 generation (Fig. 7b, c). Altogether, it appears that while preventing embryonic lipid endowment may promote adult survival post-CS, offspring that go on to experience future inclement conditions suffer diminished survival rates, underscoring the evolutionary advantage of terminal investment as a reproductive strategy.

Source: Ecology - nature.com