ABSTRACT
Oxytocin, has various physiological functions that have been well studied and many that remain unknown. Here, we aimed to determine new physiological functions of oxytocin using Caenorhabditis elegans. Oxytocin treatment promoted the restoration of movement after heat stress and enhanced the viability under heat stress. However, oxytocin had no effect on the life span and only little effect on the oxidative stress tolerance. In contrast, oxytocin treatment didn’t promote the restoration of movement or enhance the viability of deficient mutants of ntr-1/2, which is the gene encoding the oxytocin receptor. In addition, for mutants of daf-16, daf-2, tax-4, and some insulin-like peptides, the heat stress tolerance effect by oxytocin was canceled. Furthermore, oxytocin increased the expression levels of the DAF-16 target genes. Our results suggest that oxytocin treatment promoted the heat stress tolerance of C. elegans via the insulin/IGF-1 signaling pathway.
Oxytocin promotes heat stress tolerance via IIS pathway and NTR-1/2 to activate DAF-16 and increase its target genes and decreases reactive oxygen species.
Oxytocin is a neuropeptide consisting of nine amino acids [1]. This hormone has been the focus of research studies for a long time, and it is the first neuropeptide to be successfully biosynthesized [2]. In humans, oxytocin is secreted by the neurohypophysis and acts on receptors expressed in the central and peripheral nervous systems [3]. In particular, lactation and uterine contractions are well known physiological functions of oxytocin [4]. Recent studies suggested that oxytocin also has effects on human emotions, such as anti-anxiety and smoothing communication [5,6]. In addition, oxytocin treatment has been shown to promote confidence and relieve autistic behavior [7,8]. Although many studies have been conducted on the physiological functions of oxytocin, some functions may not have yet been clarified.
Oxytocin is a classical neuropeptide with high compatibility between many species, not only in mammals, but also in invertebrates such as Caenorhabditis elegans and Drosophila melanogaster [9]. In particular, C. elegans is a suitable model for the analysis of life span and the stress response, because it reflects many other animal species that have oxytocin-like peptides and it is used for analyzing the physiological function of many bioactive substances [10–14]. For example, in a previous study, treatment with acetylcholine and octopamine promoted the stress tolerance of C. elegans [14]. The study exhibited the existence of an unknown function of certain neurotransmitters. For these reasons, we aimed to determine new physiological functions of oxytocin, using the simple model organism C. elegans.
Stress tolerance and life span are promoted by DAF-16, which is a forkhead transcription factor and homologue of human FoxO [15]. DAF-16 has many target genes for stress tolerance, such as hsp-12.6, mtl-1, and sip-1 [16]. Therefore, DAF-16 plays an important role in heat stress tolerance [17]. Furthermore, chronic activation of DAF-16 in the intestine prolongs the lifespan of nematodes [16]. DAF-16 is negatively regulated via phosphorylation by AKT kinase [18]. AKT kinase is in turn activated by DAF-2, which is expressed in the cell membrane [19]. DAF-2, the ortholog of the human insulin receptor, binds to insulin like peptides (ILPs) secreted by neurons [19,20]. The secretion of ILPs is regulated by the neural Ca ion channel TAX-4 [21]. This study shows that oxytocin significantly promotes heat stress tolerance via the insulin/IGF-1 signal (IIS) pathway.
Material and methods
Strain and culture
Wild type (WT) C. elegans Bristol N2 and deficient C. elegans mutants of daf-16 (mgDf50) and daf-2 (e1370) were provided by the Caenorhabditis Genetics Center (CGC, MN, USA). Deficient mutants of ntr-1 (tm2765), ntr-2 (tm2243), ins-1 (tm1888), ins-4 (tm3620), ins-7 (tm2001), ins-11 (tm1053), ins-26 (tm1983), ins-30 (tm7056), daf-28 (tm2308), tax-4 (tm3062), unc-13 (tm5530), and unc-31 (tm5467) were provided by the National BioResource Project (NBRP, Shizuoka, Japan). Each strain was cultured on nematode growth medium (NGM) agar plates seeded with Escherichia coli OP50 (CGC).
Age-synchronization
To synchronize the growth of C. elegans, adult worms were treated with NaClO solution (NaClO [Haiter, KAO, Tokyo, Japan]:distilled water [5:1]). The eggs were cultured in S-basal medium (0.1 M NaCl, 50 mM potassium phosphate buffer [pH 6.0]) at 20°C until hatching.
Thrashing movement assay under heat stress
Age-synchronized L1 larvae were transferred onto NGM plates seeded with OP50 solution containing oxytocin (1, 5, 10 μM) (WAKO, Osaka, Japan) (Figure 1) and cultured at 20°C for 4 days. Then, adult worms were transferred to NGM plates without food and subjected to heat at 35°C for 4 hours. The worms were then transferred to an OP plate and incubated at 20°C for 12 h. Then, nematodes were transferred back to the S-basal medium and their motility was measured for 15 seconds. Whole body movement recovery was calculated by dividing the motility of the heat treatment group by that of the control group, which did not undergo heat treatment. Standard observation time points were 12 and 24 h. For deficient mutants of ntr-1/2, daf-28, and ins-7, the observation time point was 6 h; daf-2 and tax-4, 3 h.
Oxytocin promotes heat stress tolerance.
(a) Age-synchronized L1 larvae were transferred onto NGM plates seeded with OP50 solution containing oxytocin and cultured at 20°C for 4 days. Worms were treated at 35°C for 4 h. After breeding at 20°C for 12 h, their motility was measured as the number of whole body movements in 15 seconds in S-basal medium. Values on the vertical axis show exercise recovery ([momentum at 35°C/momentum at 20°C] × 100), and the horizontal axis shows the concentration of oxytocin used. n = 10, Mean ± SEM, * p < 0.05. ** p < 0.01. (b) Effect of oxytocin on viability. Age-synchronized L1 larvae were transferred onto NGM plates seeded with OP50 solution containing oxytocin and cultured at 20°C for 4 days. Viability was measured every 2 h beginning 10 h after the initiation of 35°C heat treatment. The vertical axis shows the viability, and the horizontal axis shows the survival time. n = 40, * p < 0.05, ** p < 0.01. (c) Effect of oxytocin on oxidative stress tolerance. Age-synchronized L1 larvae were transferred onto NGM plates seeded with OP50 solution containing oxytocin and cultured at 20°C for 4 days. The worms were then transferred to a 24-well plate for 0.1%H2O2 treatment. Viability was measured every 2 h. The vertical axis shows viability, and the horizontal axis shows survival time. n = 24. The graph shows the mean ± standard error. (d) Lifespan analysis. Synchronized nematodes (adult) were given oxytocin with OP50, and the viability was measured every 2 days. n = 60. The vertical axis shows the viability, and the horizontal axis shows the survival time.
Oxytocin promotes heat stress tolerance.
(a) Age-synchronized L1 larvae were transferred onto NGM plates seeded with OP50 solution containing oxytocin and cultured at 20°C for 4 days. Worms were treated at 35°C for 4 h. After breeding at 20°C for 12 h, their motility was measured as the number of whole body movements in 15 seconds in S-basal medium. Values on the vertical axis show exercise recovery ([momentum at 35°C/momentum at 20°C] × 100), and the horizontal axis shows the concentration of oxytocin used. n = 10, Mean ± SEM, * p < 0.05. ** p < 0.01. (b) Effect of oxytocin on viability. Age-synchronized L1 larvae were transferred onto NGM plates seeded with OP50 solution containing oxytocin and cultured at 20°C for 4 days. Viability was measured every 2 h beginning 10 h after the initiation of 35°C heat treatment. The vertical axis shows the viability, and the horizontal axis shows the survival time. n = 40, * p < 0.05, ** p < 0.01. (c) Effect of oxytocin on oxidative stress tolerance. Age-synchronized L1 larvae were transferred onto NGM plates seeded with OP50 solution containing oxytocin and cultured at 20°C for 4 days. The worms were then transferred to a 24-well plate for 0.1%H2O2 treatment. Viability was measured every 2 h. The vertical axis shows viability, and the horizontal axis shows survival time. n = 24. The graph shows the mean ± standard error. (d) Lifespan analysis. Synchronized nematodes (adult) were given oxytocin with OP50, and the viability was measured every 2 days. n = 60. The vertical axis shows the viability, and the horizontal axis shows the survival time.
Measurement of viability under heat stress
Age-synchronized L1 larvae were transferred onto NGM plates seeded with OP50 solution containing oxytocin and cultured at 20°C for 4 days. Then, adult worms were transferred to an OP plate containing ampicillin and maintained in a constant temperature bath at 35°C. Survival rate was measured 10 h after the heat treatment was started and every 2 h after that. To confirm survival, worms were poked with platinum wire and those without stimulus response were deemed dead.
Measurement of viability under hydrogen peroxide
Age-synchronized L1 larvae were transferred onto NGM plates seeded with OP50 solution containing oxytocin and cultured at 20°C for 4 days. To a 24-cell plate, 500 μL of 0.1% H2O2 (Sigma, Tokyo, Japan) was added, and one nematode was placed in each cell. Thereafter, the survival rate was measured every hour, as described above.
Life span assay
Age-synchronized L1 larvae were transferred onto NGM plates seeded with OP50 and cultured at 20°C for 3 days. The worms were exposed to 0.5 mg/mL 5-fluoro-2′-deoxyuridine (FUdR, WAKO) to prevent the generation of progenies and cultured at 20°C. After 1 day, 60 worms were transferred onto NGM plates dribbled with 100 mL of OP50 solution containing oxytocin and cultured at 20°C (20 worms/plate). Worms were additionally exposed to 0.5 mg/mL FUdR every 2 days from Day 0 to Day 8. Surviving worms were counted every 2 days. Thereafter, the survival rate was measured every hour, as described above.
Measurement of ROS level
Age-synchronized L1 larvae were transferred onto NGM plates seeded with OP50 solution containing oxytocin and cultured at 20°C for 4 days. Then, the worms were treated with 50 μM 2′, 7′-dichlorofluorescein (WAKO). Finally, the worms were washed and treated with 4% paraformaldehyde and observed and photographed using a BZ8000 fluorescence microscope (KEYENCE Japan, Osaka, Japan). The fluorescence intensity was measured using Image J software.
qRT-PCR
Age-synchronized L1 larvae were transferred onto NGM plates seeded with OP50 solution containing oxytocin and cultured at 20°C for 4 days. RNA was purified from whole-cell extracts of worms, using RNAiso PLUS (Takara, Shiga, Japan). cDNA was synthesized using the PrimeScript® RT reagent kit with gDNA Eraser (Takara). cDNA was amplified using the Thermal Cycler Dice® Real Time System Lite (Takara) and Thunderbird SYBR qPCR Mix (TOYOBO, Osaka, Japan). qPCR was performed by ABI 7300 with the default cycling condition [50°C/2 min, 95°C/10 min, (95°C/15 s, 60°C/min) × 40]. Actin was used as the internal control. The primers are shown in Table. 1
Statistical analysis
Statistical significance for the survival curves was analyzed using the log-rank test, and others were assessed using t-tests, with statistically significant differences defined at *p < 0.05 and **p < 0.01.
Results
Physiological function analysis of oxytocin
After breeding the nematodes at 35°C for 4 h, their movement decreased significantly, but was gradually recovered with time (12, 24 h). In contrast, the decreased movement after heat stress was significantly restored by oxytocin treatment (Figure 1(a)). Additionally, the survival rate under heat stress was significantly increased based on the dose of oxytocin administered (Figure 1(b)). The survival rate under oxidation stress also showed a similar increasing trend, but was not statistically significant (Figure 1(c)). On the other hand, oxytocin had no effect on the life span of nematodes (Figure 1(d)). Finally, oxytocin significantly decreased the ROS levels of the nematodes (Figure 2).
Oxytocin decreased ROS level.
Age-synchronized L1 larvae were transferred onto NGM plates seeded with OP50 solution containing oxytocin and cultured at 20°C for 4 days. Then, worms were treated with 50 μM DCF-DA. The worms were washed and treated with 4% PFA. (a) Scale bar indicates 600 μm. (b) The vertical axis represents the relative fluorescence intensity, calculated with CT as 1. n = 20. The graph shows the mean ± standard error. ** p < 0.01.
Oxytocin decreased ROS level.
Age-synchronized L1 larvae were transferred onto NGM plates seeded with OP50 solution containing oxytocin and cultured at 20°C for 4 days. Then, worms were treated with 50 μM DCF-DA. The worms were washed and treated with 4% PFA. (a) Scale bar indicates 600 μm. (b) The vertical axis represents the relative fluorescence intensity, calculated with CT as 1. n = 20. The graph shows the mean ± standard error. ** p < 0.01.
Action mechanism analysis of oxytocin in the mutants
Next, the mechanism by which oxytocin imparts heat stress tolerance was analyzed. The standard time points of thrashing assay are 0, 12 and 24 hours [14] (Figure 1(a)). However, daf-2 mutants restored faster than wild type worms [14]. In this study, some mutants, except daf-16, restored faster than wild type worms. Thus, thrashing were observed in 3 or 6 hours, except daf-16 mutants. The first targets were NTR-1 and NTR-2, which are oxytocin receptors of C. elegans [22,23]. After breeding deficient mutants of ntr-1 and ntr-2 at 35°C for 4 h, the movement decreased conspicuously, but was gradually recovered with time (6 h). Moreover, this decreased movement was not restored by oxytocin treatment (Figure 3(a)). The next target was DAF-16, which plays a role in stress tolerance [15–17]. After breeding deficient mutants of daf-16 at 35°C for 4 h, the movement decreased conspicuously, did not recover with time (12, 24 h), and was not restored by oxytocin treatment (Figure 3(b)). Effect of oxytocin on thrashing in 24 h was masked because CT and OX might reach to almost plateau level. DAF-16 is regulated by the IIS pathway [18–21]. After breeding deficient mutants of daf-2 and tax-4 at 35°C for 4 h, the movement decreased conspicuously, gradually recovered with time (3 h), but was not restored by oxytocin treatment (Figure 3(c, d)). After breeding deficient mutants of daf-28 and ins-7 at 35°C for 4 h, the movement decreased conspicuously, gradually recovered with time (6 h), but was not restored by oxytocin treatment (Figure 3(e)). But effects of oxytocin on wild type worms also disappeared (Figure 3(c-e)). It seem that oxytocin restored thrashing movement most efficiently in 12 h after heat stress. It is suggested that heat stress tolerance induced by oxytocin treatment is regulated by IIS pathway. However, the above evidences were not enough to conclude it because wild type worm did not show restored movement in short time (Figure 3(c-e)). Thus, the survival rate under heat stress was also analyzed using the above-mentioned mutants. When breeding deficient mutants of daf-16, daf-2, ntr-1, ntr-2, daf-28, ins-7, and tax-4 at 35°C, the survival rate was decreased when compared with wild type worms, and oxytocin treatment had no effect on this decrease in the deficient mutant (Figure 4(a-d)). Some ILPs act as antagonists of DAF-2 [24] and were therefore also analyzed. Oxytocin treatment had no effect on deficient mutants of ins-1, ins-4, and ins-30 but significantly increased the survival rate of deficient mutants of ins-11 and ins-26 (Figure 5).
Heat stress tolerance (motility) by oxytocin treatment may depend on NTR-1, NTR-2, and the IIS pathway.
Age-synchronized L1 larvae were transferred onto NGM plates seeded with OP50 solution containing oxytocin and cultured at 20°C for 4 days. Worms were treated at 35°C for 4 h. After breeding at 20°C for 3, 6 or 12 h, the motility of deficient mutants of (a) ntr-1, ntr-2, (b) daf-16, (c) daf-2, (d) tax-4, (e) daf-28 and ins-7 were measured as the number of whole body movements in 15 seconds in S-basal medium. Values on the vertical axis show exercise recovery ([momentum at 35°C/momentum at 20°C] × 100), and the concentration of oxytocin used was 10 μM. n = 10, Mean ± SEM, * p < 0.05.
Heat stress tolerance (motility) by oxytocin treatment may depend on NTR-1, NTR-2, and the IIS pathway.
Age-synchronized L1 larvae were transferred onto NGM plates seeded with OP50 solution containing oxytocin and cultured at 20°C for 4 days. Worms were treated at 35°C for 4 h. After breeding at 20°C for 3, 6 or 12 h, the motility of deficient mutants of (a) ntr-1, ntr-2, (b) daf-16, (c) daf-2, (d) tax-4, (e) daf-28 and ins-7 were measured as the number of whole body movements in 15 seconds in S-basal medium. Values on the vertical axis show exercise recovery ([momentum at 35°C/momentum at 20°C] × 100), and the concentration of oxytocin used was 10 μM. n = 10, Mean ± SEM, * p < 0.05.
Heat stress tolerance (viability) by oxytocin treatment depends on NTR-1, NTR-2, and the IIS pathway.
Age-synchronized L1 larvae were transferred onto NGM plates seeded with OP50 solution containing oxytocin and cultured at 20°C for 4 days. The viability of deficient mutants of (a) ntr-1, ntr-2, (b)daf-16, daf-2 (c)tax-4, (d)daf-28 and ins-7 were measured every 2 h beginning 10 h after the initiation of 35°C heat treatment. The vertical axis shows the viability, and the horizontal axis shows the survival time. n = 40, * p < 0.05, ** p < 0.01.
Heat stress tolerance (viability) by oxytocin treatment depends on NTR-1, NTR-2, and the IIS pathway.
Age-synchronized L1 larvae were transferred onto NGM plates seeded with OP50 solution containing oxytocin and cultured at 20°C for 4 days. The viability of deficient mutants of (a) ntr-1, ntr-2, (b)daf-16, daf-2 (c)tax-4, (d)daf-28 and ins-7 were measured every 2 h beginning 10 h after the initiation of 35°C heat treatment. The vertical axis shows the viability, and the horizontal axis shows the survival time. n = 40, * p < 0.05, ** p < 0.01.
Heat stress tolerance (viability) by oxytocin treatment depends on ILPs.
Age-synchronized L1 larvae were transferred onto NGM plate seeded with OP50 solution containing oxytocin and cultured at 20°C for 4 days. The viability of deficient mutants of (a) ins-1, ins-26, (b) ins-4, ins-11 and ins-30 were measured every 2 h beginning 10 h after the initiation of 35°C heat treatment. The vertical axis shows the viability, and the horizontal axis shows the survival time. n = 40. * p < 0.05. ** p < 0.01.
Heat stress tolerance (viability) by oxytocin treatment depends on ILPs.
Age-synchronized L1 larvae were transferred onto NGM plate seeded with OP50 solution containing oxytocin and cultured at 20°C for 4 days. The viability of deficient mutants of (a) ins-1, ins-26, (b) ins-4, ins-11 and ins-30 were measured every 2 h beginning 10 h after the initiation of 35°C heat treatment. The vertical axis shows the viability, and the horizontal axis shows the survival time. n = 40. * p < 0.05. ** p < 0.01.
The final targets were the membrane transporters; UNC-31 is the membrane transporter of ILPs and UNC-13 is the membrane transporter of neuropeptides [25,26]. When breeding wild type worms under 35°C, oxytocin increased their survival rate; however, it had no effect on deficient mutants of unc-31 and unc-13 (Figure 6).
Heat stress tolerance (viability) by oxytocin treatment depends on UNC-31 and UNC-13.
Age-synchronized L1 larvae were transferred onto NGM plates seeded with OP50 solution containing oxytocin and cultured at 20°C for 4 days. The viability of deficient mutants of (a) unc-31 and (b) unc-13 were measured every 2 h beginning 10 h after the initiation of 35°C heat treatment. The vertical axis shows the viability, and the horizontal axis shows the survival time. n = 40, * p < 0.05.
Heat stress tolerance (viability) by oxytocin treatment depends on UNC-31 and UNC-13.
Age-synchronized L1 larvae were transferred onto NGM plates seeded with OP50 solution containing oxytocin and cultured at 20°C for 4 days. The viability of deficient mutants of (a) unc-31 and (b) unc-13 were measured every 2 h beginning 10 h after the initiation of 35°C heat treatment. The vertical axis shows the viability, and the horizontal axis shows the survival time. n = 40, * p < 0.05.
Gene expression change by oxytocin treatment
The previous results suggest that DAF-16 participates in the mechanism of action of oxytocin. Thus, the mRNA expression of DAF-16 target genes, namely, hsp-12.6, mtl-1, sip-1, sod-2, and sod-5, were analyzed by qRT-PCR, as previously described [16,17,27]. The expression of these genes was increased by oxytocin (Figure 7(a)). Similar to DAF-16, HSF-1 is regulated by the IIS pathway [28]. Hence, the mRNA expression of hsp-16.2, hsp-70, and cyp-35B1, which are HSF-1 target genes [29,30], was analyzed and found to be increased by oxytocin (Figure 7(b)). Decreased expression levels of daf-28 and ins-7 are required for the activation of DAF-16 [15–19]. As the result, oxytocin decreased the mRNA levels of daf-28 and ins-7 (Figure 7(c)).
Gene expression change by oxytocin treatment.
Age-synchronized L1 larvae were transferred onto NGM plates seeded with OP50 solution containing oxytocin and cultured at 20°C for 4 days. Then, RNA was extracted and cDNA was synthesized. mRNA expression of (a) hsp-12.6, mtl-1, sip-1, sod-2, sod-5, (b) hsp-16.2, hsp-70, cyp-35B1, (c) daf-28 and ins-7 were analyzed by quantitative RT-PCR. The vertical axis represents the relative mRNA level, calculated with CT as 1. Mean ± SEM, * p < 0.05, ** p < 0.01.
Gene expression change by oxytocin treatment.
Age-synchronized L1 larvae were transferred onto NGM plates seeded with OP50 solution containing oxytocin and cultured at 20°C for 4 days. Then, RNA was extracted and cDNA was synthesized. mRNA expression of (a) hsp-12.6, mtl-1, sip-1, sod-2, sod-5, (b) hsp-16.2, hsp-70, cyp-35B1, (c) daf-28 and ins-7 were analyzed by quantitative RT-PCR. The vertical axis represents the relative mRNA level, calculated with CT as 1. Mean ± SEM, * p < 0.05, ** p < 0.01.
Discussion
Previous studies have shown that oxytocin in C. elegans acts on reproduction and learning [22,23]. The present study revealed the stress tolerance effect and the molecular mechanism that occur to nematodes by oral administration of oxytocin. This study focused on stress tolerance and life span as the physiological functions of oxytocin in these organisms. Treatment with E. coli OP50 and oxytocin promoted the restoration of movement after heat stress and enhanced the survival rate under heat stress (Figure 1(a, b)). These are novel physiological function by treatment of oxytocin. Although it was not statistically significant, oxytocin did promote the survival rate even under oxidative stress (Figure 1(c)). Furthermore, oxytocin decreased the ROS levels in C. elegans (Figure 2). ROS is one of the major factors of aging [31]. Therefore, it was predicted that oxytocin prolongs the life span of nematodes. However, oxytocin had no actual effect on the life span (Figure 1(d)). These results suggest that oxytocin only promotes stress tolerance, but has no effect on chronic physiological functions such as life span, under these experimental conditions.
Then, the mechanism of heat stress tolerance was analyzed. Results obtained using gene deficient mutants showed that oxytocin promotes heat stress tolerance via NTR-1 and NTR-2 (Figures 3(a) and 4(a)). In other words, oxytocin that treated to nematodes affected via receptor. In addition, deficient mutants of ntr-1/2 showed higher viability and recovery of motility than wild type worms. It is possible that NTR-1 complemented NTR-2 activity in deficient mutant of ntr-2 and vice versa. Furthermore, the number of egg-lying decreased by lack of ntr-2 (data not shown). Thus, it is suggested that trade-off, not stress tolerance, may promoted basal viability of deficient mutant of ntr-2 under the heat stress [32]. In addition, DAF-16 is one of the transcription factors that regulate the stress tolerance of nematodes and it is regulated by the IIS pathway [15,19,20]. Oxytocin did not promote the heat stress tolerance of deficient mutants of daf-16, daf-2, daf-28, ins-7, and tax-4. These results showed that the physiological function of oxytocin depends on the IIS pathway (Figures 3(b-e) and 4(b-d). Some ILPs, namely, INS-1, INS-4, INS-11, INS-26, and INS-30, act as antagonists of DAF-2 [24]. Based on the results of using deficient mutants of these genes, the function of oxytocin depends on INS-1, INS-4, and INS-30 but not on INS-11 and INS-26 (Figure 5). Furthermore, oxytocin also depends on UNC-31 (Figure 6(a)), which is a membrane transporter of ILPs [25].
In C. elegans, major ILPs are secreted by head neurons, such as the ASI and ASJ neurons [19]. However, NTR-1 and NTR-2 are not expressed on ASI/ASJ neurons [22,23]. INS-1 is expressed on the ASH neuron, which expresses NTR-1/2 [33–35]. However, our results showed that multiplex ILPs are required for the function of oxytocin (Figures 4(b-d) and 5). These results suggest that some interactions exist between the neuron where oxytocin receptor expressed and ASI/ASJ neurons. The transport of neuropeptides between neurons is mediated by UNC-13 [26]. Our results showed that oxytocin had failed to effect on the deficient mutant of unc-13 (Figure 6(b)). This result suggested that neuron expressed NTR-1/2 sent some signal to ASI/ASJ neuron to regulate secretion of ILPs.
Finally, mRNA expression levels were analyzed. Oxytocin treatment increased the expression levels of heat stress tolerance genes such as hsp-12.6, mtl-1, and sip-1 (Figure 7(a)). Furthermore, the expression of sod-2 and sod-5 was also increased by oxytocin (Figure 7(a)), suggesting that oxytocin decreased the ROS levels via these genes. HSF-1 is also negatively regulated by the IIS pathway [28–30], and HSF-1 interacts with DAF-16 in response to heat stress [16,36]. Our results showed that oxytocin treatment increased the expression levels of HSF-1 target genes such as hsp-16.2, hsp-70, and cyp-35B1 (Figure 7(b)). Furthermore, the expression levels of daf-28 and ins-7 were decreased (Figure 7(c)). The series of mRNA expression analyses suggest that oxytocin promotes the transcriptional activity of DAF-16 and HSF-1 via decreasing daf-28 and ins-7.
Conclusion
It was revealed that administration of oxytocin to nematodes enhances resistance to heat stress. NTR-1/2 and insulin signal were involved in its action, which was caused by activation of DAF-16 and HSF-1. The present findings found unknown physiological effects of oxytocin and opened up the possibility of new pharmacological application to mammals including humans.
Acknowledgments
This work was supported in part by Grants-in-Aid for Scientific Research and Education from the University of Tsukuba, Japan.
Conflict of interest statement
On behalf of all authors, the corresponding author states that there is no conflict of interest.
Author Contributions
KY and KS conceived and designed the study, KY performed experiments, KS provided essential tools and reagents, KY and KS analyzed data, KY and KS wrote the paper. KY and KS made manuscript revisions. KS supervised the study as a principal investigator. All authors read and approved the final manuscript.
Disclosure statement
There are no conflicts of interest to declare.
