Self-sperm induce resistance to the detrimental effects of sexual encounters with males in hermaphroditic nematodes
Animals interact with each other in complex ways that can affect their health , including sexual , cooperative , and competitive interactions . Sexual interactions drastically impact an individual’s health and behavior . In Drosophila and C . elegans , sexual interactions are detrimental to health and shorten the lifespan of both sexes ( Aprison and Ruvinsky , 2016; Chapman et al . , 1995; Fowler and Partridge , 1989; Gems and Riddle , 1996; Gendron et al . , 2014; Harvanek et al . , 2017; Maures et al . , 2014; Partridge and Farquhar , 1981; Promislow , 2003; Promislow , 1992; Shi and Murphy , 2014; Shi et al . , 2017; Ting et al . , 2014; Van Voorhies , 1992; Woodruff et al . , 2014; Wu et al . , 2012 ) . In mammals , mates induce neurological , developmental , and behavioral changes in the opposite sex and can negatively impact health ( Gao et al . , 2017; Garratt et al . , 2016; Aloise King et al . , 2013; Stowers and Kuo , 2015 ) . For example , the presence of males can increase female body weight ( Garratt et al . , 2016 ) and accelerate puberty ( Flanagan et al . , 2011; Vandenbergh , 1969 ) . Identification of specific strategies involved in responding to the effects of sexual encounters could help to improve our understanding of how the sexes interact with each other , how sexual pressures have shaped species over evolutionary time , and how these mechanisms could be harnessed to improve health . C . elegans is particularly well suited to the study of the effect of sexual interactions on lifespan . Males shorten the lifespan of the C . elegans hermaphrodite through a phenomenon called male-induced demise ( Gems and Riddle , 1996; Maures et al . , 2014; Shi and Murphy , 2014 ) . Previous studies showed that males shorten hermaphrodite lifespan by several means ( e . g . sperm , seminal fluid , and pheromones ) , and identified some of the hermaphrodite genes that mediate male-induced demise ( e . g . utx-1 , ins-11 , daf-16 ) ( Maures et al . , 2014; Shi and Murphy , 2014 ) . However , whether hermaphrodites evolved natural defense mechanisms to protect themselves after a sexual encounter with a male remained unknown . Here we discover that young hermaphrodites are entirely protected from brief sexual encounters with males . Surprisingly , the natural protection in young hermaphrodites is not due to their youthfulness , but rather to the presence of self-sperm . Self-sperm act through a sperm-sensing pathway to protect the soma in a fertilization-independent manner , and activating this pathway in females can protect them against the detrimental effect of males . The protective effect of self-sperm is conserved in other hermaphroditic nematodes and may represent a key adaptation for their reproductive success .
Previous studies were done with long encounters between young C . elegans hermaphrodites and males ( Gems and Riddle , 1996; Maures et al . , 2014; Shi and Murphy , 2014 ) , which does not reflect the situation in nature where males are rare ( Barrière and Félix , 2005 ) . We thus asked if varying the length of sexual interactions , as well as the age of males and hermaphrodites , could reveal natural strategies that evolved to mitigate the negative impact of sexual encounters on health ( Figure 1A ) . As previously shown ( Gems and Riddle , 1996; Maures et al . , 2014; Shi and Murphy , 2014 ) , long and frequent sexual interactions with young males shortened hermaphrodite lifespan ( Figure 1B and Figure 1—figure supplement 1A ) . Middle-aged males were less able to induce premature demise of the opposite sex than young males ( Figure 1B ) , probably because of their decreased ability to efficiently mate ( Guo et al . , 2012 ) ( Supplementary file 3 ) . To better mimic the natural situation—where sexual interactions are infrequent due to the rarity of males—and isolate the mating-specific aspect of sexual interactions , we tested how a brief ( two-hour ) mating with young males impacts young and middle-aged hermaphrodites ( Figure 1C , D ) . Because the chance of a sexual interaction is relatively low in the brief two-hour exposure and can differ depending on the age of the hermaphrodite ( Supplementary file 3; Garcia et al . , 2007; Leighton et al . , 2014 ) , we specifically measured the lifespans of hermaphrodites that mated using a fluorescent sperm tracking method to isolate hermaphrodites that received male sperm ( Figure 1E and Figure 1—figure supplement 1B–F ) ( Stanfield and Villeneuve , 2006 ) . Interestingly , while a brief encounter with males induced the premature death of middle-aged hermaphrodites , it did not affect the lifespan of young hermaphrodites ( Figure 1C , D , F and G ) . Young hermaphrodites were remarkably resistant to the negative impact of males , even though they successfully mated ( Figure 1C , F ) . We confirmed this observation using two different male strains and two methods of isolating mated hermaphrodites ( Figure 1C , D , F and G ) . Thus , shortening the length of sexual interactions , which is more similar to the situation in nature , reveals that young hermaphrodites can defend themselves against the lifespan-shortening effect of mating with males . Young and middle-aged hermaphrodites differ not only by their ages , but also in their reproductive status . Young hermaphrodites are self-fertile , due to the presence of both oocytes and self-sperm . In contrast , middle-aged hermaphrodites are no longer self-fertile , because they have exhausted their self-sperm , though they can still reproduce if their oocytes are fertilized by males ( Riddle et al . , 1997 ) . To disentangle age and reproductive status , we compared the lifespan of young hermaphrodites to that of young ‘feminized’ individuals ( which have oocytes , but no self-sperm; Ellis and Schedl , 2007 ) after a brief encounter with males . Surprisingly , young feminized individuals ( fem-1[hc17] or fog-2[q71]; Doniach and Hodgkin , 1984; Schedl and Kimble , 1988 ) were sensitive to mating-induced death and exhibited premature death and deterioration ( Figure 2A–D and Figure 2—figure supplement 1A , B ) . Although mating efficiency can vary between C . elegans mutants and individuals of different ages ( Supplementary file 3; Garcia et al . , 2007; Leighton et al . , 2014; Morsci et al . , 2011 ) , we specifically measured the lifespan of only the hermaphrodites that mated ( i . e . received male sperm ) ( Figure 1E ) . Thus , mating efficiency differences are unlikely to contribute to the sensitivity of feminized individuals to males . Consistent with the observation that the presence of self-sperm is necessary to live a normal lifespan even after a brief encounter with males , young individuals that lack self-sperm and oocytes ( glp-1[e2144]; Priess et al . , 1987 ) also lived shorter after a brief encounter with males ( Figure 2—figure supplement 1C , D ) , even though glp-1 mutants are normally long-lived ( Antebi , 2013; Arantes-Oliveira et al . , 2002; Hansen et al . , 2013; Keith and Ghazi , 2015; Wang et al . , 2008 ) . Together , these results indicate that a major factor in protecting an individual from the detrimental influence of males is not youthfulness , but the presence of self-sperm . To further investigate the importance of self-sperm in the resistance of young hermaphrodites to the negative impact of mating with males , we performed RNA-seq on young or middle-aged hermaphrodites or feminized individuals that were never exposed to males ( no males ) or had a brief encounter with males ( +male sperm ) ( Figure 2—figure supplement 2A ) . Principal Component Analysis ( PCA ) revealed that , as expected ( Angeles-Albores et al . , 2017 ) , the transcriptomes of hermaphrodites and feminized individuals that were never exposed to males separated based on fertility status and , to some extent , age ( Figure 2E , F and Figure 2—figure supplement 2B ) . Interestingly , the first principal component separated the transcriptomes of hermaphrodites that have a shortened lifespan ( middle-aged upon mating ) from those of hermaphrodites that live a normal lifespan ( young upon mating , young and middle-aged without mating ) ( Figure 2E ) . Consistently , the first principal component also separated the transcriptomes of feminized individuals that have a shortened lifespan ( young and middle-aged upon mating ) from those of feminized individuals that live a normal lifespan ( young and middle-aged without mating ) ( Figure 2F ) . This observation confirms the importance of self-sperm in the protection from males . GO terms linked with individuals that are resistant ( e . g . young hermaphrodites ) or sensitive to mating-induced death ( e . g . young feminized ) included translation , lipid metabolism , the innate immune response , and protein homeostasis ( proteostasis ) ( Figure 2G–I , Figure 2—figure supplement 2C , and Figure 2—source data 1 and 2 ) , suggesting these conserved homeostatic pathways could be responsible for the lifespan differences in the response to males . Indeed , many of these pathways are linked with longevity ( Kaeberlein et al . , 2015; Kenyon , 2010; López-Otín et al . , 2013; Riera et al . , 2016; Shore and Ruvkun , 2013 ) . Collectively , these data indicate that the presence of self-sperm is necessary to protect the soma against deterioration due to brief encounters with males , perhaps by regulating homeostasis pathways . Self-sperm play a key role in germline quality assurance in C . elegans by triggering the clearance of carbonylated and aggregated proteins from the germline ( Bohnert and Kenyon , 2017; Goudeau and Aguilaniu , 2010 ) . However , whether self-sperm could harness this potential to protect the soma is not known . To determine if the presence of self-sperm is sufficient to protect hermaphrodites , we examined the response of individuals with a masculinized germline ( which have a female soma and self-sperm , but no oocytes ) , fem-3 ( q20 ) ( Ahringer and Kimble , 1991; Barton et al . , 1987; Ellis and Schedl , 2007 ) . Masculinized individuals that only have self-sperm were protected from premature death induced by mating with males not only when young , but even at an older age when hermaphrodites normally become sensitive to an encounter with males ( Figure 3A–D , and Figure 3—figure supplement 1A , B ) . These data suggest that the presence of self-sperm is sufficient to protect from the detrimental effects on lifespan of a brief mating with males . We next asked whether self-sperm could act via their ability to self-fertilize hermaphrodites . Interestingly , hermaphrodite mutants that are defective in self-fertilization despite maintaining self-sperm ( spe-9[hc88]; Ellis and Stanfield , 2014b; Singson et al . , 1998 ) were still protected from early death induced by mating with males when young ( Figure 3E , F ) . In contrast , hermaphrodites that are defective in self-sperm maturation ( due to loss of the SPE-44 transcription factor; Kasimatis et al . , 2018; Kulkarni et al . , 2012 ) were no longer protected from the lifespan shortening effects of mating with males when young ( Figure 3G and Figure 3—figure supplement 1C , D ) . Thus , the presence of mature self-sperm , but not self-fertilization , is sufficient for the protection against mating-induced demise . We next explored the mechanisms by which self-sperm protect individuals . Self-sperm could protect individuals independently of fertilization , by acting via sperm-sensing pathways . In C . elegans , sperm proteins are known to be sensed by the somatic gonad and to signal to the germline and somatic gonad by repressing the homeodomain transcription factor CEH-18 and the Ephrin receptor VAB-1 ( Figure 4A and Greenstein et al . , 1994; Miller , 2001; Miller , 2003 ) . Hence , the loss of CEH-18 and VAB-1 can mimic the presence of self-sperm ( Miller , 2003 ) . Deficiency in CEH-18 and VAB-1 was sufficient to protect middle-aged hermaphrodites that have depleted their self-sperm from the negative effects of mating with males ( Figure 4D , E ) . Interestingly , deficiency in CEH-18 and VAB-1 in young feminized individuals made them resistant to brief encounter with males ( Figure 4B , C ) , indicating that this conserved pathway could be sufficient to protect females against the negative impact of males . To understand how this pathway might work to protect the soma , we examined the potential links between the transcription factor CEH-18 and the genes differentially expressed between hermaphrodites and feminized individuals that we identified by RNA-seq . ceh-18 mRNA levels were not significantly affected by the presence of self-sperm or mating with a male ( Figure 4—figure supplement 1A ) . In contrast , several genes that interact genetically or physically with CEH-18 , including the chromatin modifiers set-33 and jmjd-3 . 2 , were differentially expressed between young hermaphrodites and feminized individuals that have successfully mated with males ( Figure 4F and Figure 4—figure supplement 1B ) . These observations suggest that differences in chromatin state between young hermaphrodites and feminized individuals may contribute to the transcriptional effect of the CEH-18 sperm-sensing transcription factor in the resistance and sensitivity to mating-induced death . A subset of the genes regulated by the presence of self-sperm or mating with a male contained a CEH-18 binding site in their regulatory regions ( Figure 4—source data 1 ) , though the CEH-18 motif and binding peaks ( Kudron et al . , 2018; Narasimhan et al . , 2015 ) were not significantly enriched in these genes ( perhaps due to cell heterogeneity ( Cao et al . , 2017 ) , see Material and methods ) . We asked if the subset of genes that are differentially expressed between young hermaphrodites and young feminized individuals and contain a CEH-18 binding site were enriched for specific biological features ( Figure 4—source data 1 and 2 ) . Interestingly , GO term enrichment of these genes revealed several terms linked with longevity , including ‘Determination of adult lifespan’ ( Figure 4G , Figure 4—figure supplement 1C ) , suggesting that the sperm-sensing transcription factor CEH-18 may directly regulate genes that affect longevity . Together , these results show that self-sperm protect individuals , even older ones , from mating-induced death by repressing a sperm-sensing pathway in the somatic gonad , which could in turn protect hermaphrodites by altering chromatin and transcriptional networks . Is the ability of self-sperm to promote resistance to brief interactions with males unique to C . elegans or a common hermaphroditic strategy for resistance to the negative impact of mating with males to optimize their reproduction and health ? The C . elegans ancestor was a gonochoric species with true females and obligatory males ( Kiontke et al . , 2004 ) . Hermaphroditism evolved at least three times independently in this lineage ( in C . elegans , C . briggsae , and C . tropicalis ) ( Ellis and Lin , 2014a; Guo et al . , 2009; Hill et al . , 2006; Kiontke et al . , 2004; Kiontke et al . , 2011; Nayak et al . , 2005; Thomas et al . , 2012 ) ( Figure 5A ) . Other nematode species remained true females ( e . g . C . remanei and C . brenneri ) ( Figure 5A ) . Both hermaphroditic and true female nematodes are known to succumb prematurely following long interactions with males ( Figure 4—figure supplement 1A , B and Maures et al . , 2014; Palopoli et al . , 2015; Shi and Murphy , 2014 ) , but the effect of brief interactions has not been tested . We found that C . remanei and C . brenneri females lived normal lifespans following brief , two-hour interactions with males at all of the ages we tested ( Figure 5B and Figure 5—figure supplement 1B ) , suggesting that the differential sensitivity of old versus young hermaphrodites may have evolved in hermaphroditic species . Indeed , we found that young C . briggsae hermaphrodites ( which have self-sperm ) resisted a brief exposure to males and lived a normal lifespan whereas middle-aged C . briggsae hermaphrodites ( which have depleted their self-sperm ) succumbed to brief encounter with males ( Figure 5C and Figure 5—figure supplement 1C ) . Interestingly , feminized individuals in C . briggsae ( due to a different mutation than C . elegans , the she-1 feminizing mutation , which affects sex determination; Guo et al . , 2009 ) , also became sensitive to a brief interaction with males when young and exhibited premature death ( Figure 5D ) . As with C . elegans , we controlled for age- and genotype-dependent differences in C . briggsae mating efficiency ( Supplementary file 3 ) using fluorescent male sperm tracking ( Figure 1E ) . Does the sperm-sensing pathway mediate self-sperm protection from mating-induced demise in C . briggsae as it does in C . elegans hermaphrodites ? We first verified that knock-down of the orthologous sperm-sensing pathway in C . briggsae females ( mfIs42[Cel-sid-2; Cel-myo-2::DsRed]; she-1[v35] ) indeed reduced the accumulation of unfertilized oocytes in the gonad arms and increased the number of unfertilized oocytes in the uterus ( Figure 5—figure supplement 3 ) similar to C . elegans ( Miller , 2003 ) . Interestingly , knock-down of the C . briggsae sperm-sensing genes protected middle-aged hermaphrodites ( which are self-sperm depleted ) from the lifespan shortening effects of a brief interaction with males ( Figure 5E , F ) . Together , these results suggest that hermaphroditism , notably the presence of self-sperm and detection by the sperm-sensing pathway , may have co-evolved more than once with strategies to protect from the detrimental effect of sexual interactions with the opposite sex . This may be particularly important to allow hermaphrodites to maximize their reproductive success by fertilizing their eggs through self-fertilization and mating with another male later in life . As male sperm out-compete hermaphroditic self-sperm ( LaMunyon and Samuel , 1999; Ward and Carrel , 1979 ) , such a strategy would also allow for the hermaphroditic self-sperm to be used . Because the sperm-sensing pathway is conserved in species with true females and is sufficient to protect feminized individuals , this pathway could be leveraged to protect the soma in a conserved manner , even in other species .
Here we show that a sensing mechanism between the germline and the soma mediates protection against the negative impact of sexual interactions . While previous work revealed the phenomenon of male-induced demise and identified specific mutations that could protect individuals from male-induced demise ( Gems and Riddle , 1996; Maures et al . , 2014; Shi and Murphy , 2014 ) , it was unknown whether natural defenses exist to protect from the detrimental effects of males and what their mechanism of action could be . The previous studies investigated the effects of prolonged interactions between the sexes . While there is evidence of occasional ‘bursts’ of high rates of males in wild C . elegans populations ( Barrière and Félix , 2005; Frézal and Félix , 2015; Sivasundar and Hey , 2005 ) , males in the wild are thought to be rare . Thus , sexual interactions in nature are likely to be quite brief . Here , we have used very brief interactions between the sexes to uncover phenomena that better reflect the natural situation . We show for the first time that innate mechanisms that involve self-sperm can protect against the deleterious effects of mating with males . The presence of self-sperm in hermaphrodites is also known to decrease attraction of and mating with males ( Garcia et al . , 2007; Leighton et al . , 2014; Morsci et al . , 2011; Shi and Murphy , 2014 ) , and we did account for this in our study by selecting only mated individuals . Hence , self-sperm may be evolutionary important to ensure , in several different ways , that hermaphrodites produce as many of their own self-progeny as possible before succumbing to mating-induced death . Whether the ability of males to shorten the lifespan of older hermaphrodites that lack self-sperm is advantageous in nature remains unknown . The number of animals that reach middle or older-age in the wild is probably low . However , the protective effect of self-sperm on the soma appears to have evolved independently in two distantly-related species , suggesting that it is important for nematode hermaphrodites . The role of self-sperm as a signal to regulate the response to the opposite sex may also have evolved in other animals , including self-fertilizing hermaphroditic species ( e . g . snails , slugs , and the vertebrate mangrove killifish ) ( Jarne and Auld , 2006; Mackiewicz et al . , 2006 ) . The ancestor of C . elegans and related nematodes did not have self-sperm-mediated protection from mating-induced death and our data indicate that self-sperm mediated protection was an evolutionary innovation in the C . elegans and C . briggsae lineages . Whether the evolution of resistance to mating-induced death in C . elegans and C . briggsae occurred as the result of linking ancestral protection mechanisms to self-sperm , or whether novel mechanisms evolved in these hermaphroditic lineages , is unknown . However , given that self-sperm mediated mating-induced death protection evolved more than once in Caenorhabditis nematodes , the most parsimonious model is that the hermaphroditic lineages linked ancestral protection mechanisms with self-sperm . Indeed , the signaling pathways that are engaged by self-sperm ( an Ephrin receptor and homeodomain transcription factor ) are conserved in nematodes , suggesting that these ancestral pathways were harnessed in unique ways in the hermaphroditic lineages to evolve self-sperm mediated resistance to mating-induced death . This raises the intriguing possibility that such protective mechanisms could also be ‘tuned up’ in female species by mimicking activation of the signaling pathways that are triggered by self-sperm . Interestingly , the Ephrin receptor that detects the presence of sperm is conserved in mammals , it is expressed in the somatic gonad ( Figure 5—figure supplement 2B; Uhlén et al . , 2015 ) , and is linked with mammalian fertility ( Barban et al . , 2016; Buensuceso et al . , 2016 ) . Thus , some of the mechanisms that signal between germline and soma could be used more generally to protect against the negative impact of sexual interactions in other species . Fertilization has a rejuvenating effect in the germline and resets the aging clock in each generation ( Rando and Chang , 2012; Unal et al . , 2011 ) . Consistently , self-sperm can clear carbonylated and aggregated proteins , two aging hallmarks , in the germline of C . elegans ( Bohnert and Kenyon , 2017; Goudeau and Aguilaniu , 2010 ) . Resetting of aging features can be achieved to some degree in mammalian somatic cells by the process of reprogramming to induced pluripotent stem cells , which can mimic fertilization and has a ‘rejuvenating’ capacity ( Rando and Chang , 2012 ) . In mammals , triggering rejuvenating factors in somatic cells , in a manner that is uncoupled from de-differentiation , has been recently suggested to be a potential rejuvenation strategy ( Ocampo et al . , 2016 ) . However , the potential role of sperm in protection , and possible rejuvenation , of the soma is not known . Given the effect of sperm and fertilization on resetting the aging clock in the germline , as well as the conservation of key elements of the sensing pathway , it is possible that the protective properties of self-sperm , or even sperm itself , are conserved in other species , including mammals . These findings could open new strategies for harnessing the potential of gametes and their sensing pathways to ‘reset’ some hallmarks of aging .
All C . elegans , C . briggsae , C . remanei , and C . brenneri WT and mutant strains used in this study are listed in Supplementary file 1 . All strains were maintained on Nematode Growth Media ( NGM ) plates with 50 μg/mL streptomycin ( Gibco ) and a lawn of OP50-1 bacteria ( a gift from M . -W . Tan ) from stationary phase cultures . Nematodes were grown at 20°C , with the exception of temperature-sensitive mutants ( glp-1[e2144] , fem-1[hc17] , fem-3[q20] , and spe-9[hc88] ) , which were maintained at 15°C ( permissive temperature ) . When temperature sensitive mutants were used for assays , they were grown at the restrictive temperature ( 25°C ) . The genotype of strains was verified by genotyping PCR and Sanger sequencing and the strains were backcrossed three times into our laboratory’s N2 strain ( in addition to the backcrossing that was performed when the mutants were initially isolated ) . Because some C . elegans strains have been recently reported to inadvertently contain a long-lived allele of the fln-2 gene ( personal communication , Y . Zhao , H . Wang , R . J . Poole and D . Gems [the Worm Breeder’s Gazette , 2018] ) , we verified the fln-2 genotype using genotyping by PCR followed by Sanger sequencing using the following primers ( 5'-GGGTGAAGAATGAGAAACACGC and 5'-ATGATGCAGTTTTGCCAACGG ) . The forward primer ( listed first ) was used for sequencing . We confirmed that the key strains used in this study—N2 ( WT ) , BA17 ( fem-1[hc17] ) , CB4108 ( fog-2[q71] ) , CF1903 ( glp-1[e2144] ) , JK816 ( fem-3[q20] ) , DG1604 ( fog-2[q71]; ceh-18[mg57] ) , and GR1034 ( ceh-18[mg57] ) —did not contain this long-lived allele of fln-2 and were wild-type for this allele . C . elegans , C . brenneri , C . remanei , and C . briggsae lifespan assays were performed in the same manner . Hermaphrodites or females for these assays were age-synchronized with a short ( 3–4 hr ) egg-lay using young ( day 3–5 of life ) , well-fed adult parents . For the female species ( C . brenneri and C . remanei ) , age-synchronized , virgin females were identified and isolated from males by placing them on fresh plates at the L4 stage . All worms were grown on NGM plates with streptomycin ( 50 μg/mL ) and seeded with OP50-1 bacteria unless RNAi knock-down was performed . In the case of RNAi knock-down , worms were cultured on NGM containing ampicillin ( 100 μg/mL , Sigma ) and IPTG ( 0 . 4 mM , Invitrogen ) . During development , worms were fed HT115 bacteria ( grown to stationary phase , RNAi expression induced for 2–4 hr with 0 . 4 mM IPTG , and the bacteria concentrated to 20x ) carrying empty vector ( EV ) . Upon adulthood ( day 3 of life ) , HT115 bacteria ( grown to stationary phase , RNAi expression induced for 2–4 hr with 0 . 4 mM IPTG , and the bacteria concentrated to 20x ) carrying the appropriate RNAi clone from the Ahringer RNAi library ( Kamath et al . , 2003 ) ( a gift from A . Fire ) or RNAi clones created in this study ( see ‘C . briggsae RNAi knock-down’ ) . The inserts of the plasmids encoding the RNAi clones used in this study were sequenced to verify their identity . For each assay , worms were scored as dead or alive and transferred to new plates daily during the reproductive period and then every other day . Worms were scored as dead if they did not respond to gentle , repeated prodding with a wire pick ( 90% Pt , 10% Ir ) along different points of their body . Worms were scored as censored if they crawled off the media or died due to bagging ( internal hatching ) or vulval rupture . Data from these censored worms were included up until the point of censorship ( see Supplementary file 2 for all data ) . For conditions in which the effect of sexual interactions was assessed , we used one of three methods , as indicated . For the long-term exposure method ( described in Maures et al . , 2014; Shi and Murphy , 2014 ) , young males ( day 1 to 2 of adulthood ) were added to the hermaphrodites at the onset of adulthood . For lifespan experiments in which the hermaphrodites were exposed to males for their entire adulthood ( Maures et al . , 2014 ) , males were added in a 1:1 ratio with hermaphrodites and the number of males remained fixed , even as hermaphrodites began to die or censored . Male worms were replaced every other day at the time the hermaphrodites were transferred to new plates . Male stocks were set up every day for the entirety of the lifespan assay . For the lifespan experiments in which hermaphrodites were exposed to males for only one day ( Shi and Murphy , 2014 ) , young males were added in a 2:1 male:hermaphrodite ratio . Following 24 hr of exposure , hermaphrodites were moved to new plates and did not encounter a male again throughout their lifespan . For the newly established , short mating-induced demise lifespan experiments , the males and hermaphrodites were only allowed to interact for 2 hr at the age specified for each assay . Males were twice as abundant as hermaphrodites during the mating period . Following a mating period of two hours , mating was assessed by the presence of fluorescent male sperm ( see below ) . These mated hermaphrodites did not interact with an adult male again and worms on plates in which male progeny reached adulthood and could have mated with the hermaphrodites were censored . Synchronized individuals ( hermaphrodites , feminized individuals etc . ) were randomly assigned to the ‘no males’ or ‘+males’ conditions by picking them onto fresh plates in an alternating manner to avoid selection bias . Similarly , the males used for mating with individuals of different genotypes or ages were from the same sets of males and were allocated randomly in an alternating manner . For each single biological replicate , approximately 35 individuals were placed on each of 2–4 plates ( each plate represents a technical replicate ) . The number of individuals per plate and number of technical replicates were chosen based on field standards ( Lucanic et al . , 2017 ) . For feminized and sterile mutants , slight modifications were made to the methods . The sterile glp-1 ( e2144 ) mutant , fertilization defective spe-9 ( hc88 ) mutant , masculinized fem-3 ( q20 ) mutant , and WT control parents were used for an egg lay at the permissive temperature ( 15°C ) and following the egg lay , the individuals used for the assay were kept at 25°C for the remainder of the assay . The feminized fem-1 ( hc17 ) mutants and the WT control parents were kept at 15°C for the egg lay and these eggs developed until day 3 of life ( adulthood ) at 25°C ( the restrictive temperature ) . Then , both the fem-1 ( hc17 ) feminized worms and the WT worms were moved to 20°C for the remainder of the assay . The lower temperature did not impact the feminized phenotype . For the auxin-inducible degradation of the transcription factor SPE-44 , the worms were grown on NGM with 1 mM auxin ( 3-indoleacetic acid , Sigma-Aldrich ) from egg lay to adult day one and were then cultured on NGM without auxin for the remainder of their lifespan . These individuals were fully self-sterile as expected ( Kasimatis et al . , 2018 ) . Finally , the C . elegans fog-2 ( q71 ) and C . briggsae she-1 ( v35 ) feminized mutants were kept at 20°C for the entire assay . For these assays , virgin feminized individuals were isolated from males at the L4 stage and were either kept away from males for the entire lifespan or only interacted with males for 2 hr as described . Lifespan data were plotted as Kaplan-Meier survival curves in Prism 7 and statistical analyses performed using the logrank ( Mantel-Cox ) test . The number of animals ( n ) used for each assay and the number of independent biological replicates ( N ) can be found in Supplementary file 2 . To identify and isolate hermaphrodites or females that have mated during a brief period of interacting with males , we modified a previously developed technique ( Stanfield and Villeneuve , 2006 ) . The day before the mating assay , adult day one males ( for C . elegans , either him-5[e1467] or nIs128[Ppkd-2::GFP]; him-8[e1469] , for C . briggsae , Cbr-him-8[v186] , and WT for C . brenneri and C . remanei ) were fluorescently labeled by culturing them overnight on NGM plates seeded with 100 μL stationary phase OP50-1 and 5 ng/μL MitoTracker Red CMXRos ( Thermo Fisher cat# M7512 , resuspended in DMSO [Fisher] at 100x and kept at −20°C in aliquots ) at a density of approximately 100 males per 6 cm plate . On the day of the mating assay , 40 fluorescently-labeled males and 20 unlabeled hermaphrodites were placed on 6 cm NGM plates seeded with OP50-1 bacteria . Animals were allowed to interact with each other and mate for 2 hr at their normal culturing conditions . Following this period , hermaphrodites that received male sperm as a result of mating were identified based on the presence of fluorescence ( male sperm ) in their uterus and/or spermatheca ( Figure 1E and Figure 1—figure supplement 1B–D ) using a fluorescent dissecting microscope or the COPAS large particle biosorter as indicated . In a small number of cases , a hermaphrodite consumed some of the MitoTracker Red CMXRos labeled bacteria resulting in red fluorescence in the gut or throughout the body . When this occurred , the individual was censored from the experiment because of the difficulty in determining if they received male sperm or not . For mating efficiency assays , 5–7 mating plates ( n ) were typically used per experimental condition and one to two independent , biological replicates ( N ) were performed . The number of mated hermaphrodites was compared to the total number of hermaphrodites per plate ( mating efficiency = # hermaphrodites with male sperm/total # hermaphrodites ) . To better understand the dynamics of mating during a two-hour period ( Figure 2—figure supplement 1E ) , we performed a separate assay using a slightly modified version of the mating efficiency method such that two male genotypes were used . Specifically , on each plate 20 young , unlabeled hermaphrodites or feminized individuals interacted with 20 unlabeled nIs128 ( Ppkd-2::GFP ) ; him-8 ( e1469 ) males and 20 MitoTracker Red CMXRos labeled him-5 ( e1467 ) males for two hours . Following this 2 hr interaction , hermaphrodites that received male sperm from the fluorescently labeled him-5 ( e1467 ) males were isolated and placed individually on 3 cm NGM plates to lay eggs . To determine if any of these him-5 ( e1467 ) mated individuals also mated with a nIs128 ( Ppkd-2::GFP ) ; him-8 ( e1469 ) male , we looked for male progeny that carried nIs128[Ppkd-2::GFP] transgene . These individuals were thus mated at least twice and received male sperm from at least two males . For lifespan assays , approximately eight mating plates were set up per condition . Hermaphrodites or females that received male sperm were moved to fresh plates without males at a density of approximately 35 individuals per plate and tracked for the remainder of their lifespan . To better understand the resistance of young hermaphrodites to mating-induced demise , we characterized the transcriptomes of the young , WT hermaphrodites that have a normal lifespan after a brief interaction with males to those that have a shortened lifespan after a brief interaction with males ( the self-sperm depleted fem-1[hc17] feminized individuals and the middle-aged , WT hermaphrodites ) . WT and fem-1 ( hc17 ) mutant individuals were age-synchronized using a brief , 3–4 hr egg lay ( see ‘Lifespan Assays’ above ) on NGM plates seeded with OP50-1 bacteria and grown at 25°C during development and the young adult stage . On day 3 of life ( egg lay is day 0 ) , the worms were all moved to 20°C for the remainder of their lifespan . This set up resulted in fully penetrant feminized germline phenotypes for the fem-1 ( hc17 ) mutants and delayed the age-associated appearance of red autofluorescence in the gut that would have made accurate detection of MitoTracker Red CMXRos labeled male sperm in the uterus and spermatheca difficult . For each of the four biological replicates , egg lays were performed on two separate days such that there was a cohort of age-synchronized young ( day 3 of life ) and middle-aged ( day 7 of life ) hermaphrodites of both genotypes on the day of mating . Following the Materials and methods section ‘Fluorescent Sperm Tracking to Identify Mated Hermaphrodites for Mating Efficiency Assays and for Lifespan Assays’ , we identified and isolated young and middle-aged WT and fem-1 ( hc17 ) individuals that received male sperm from fluorescently labeled him-5 ( e1467 ) males and transferred them to fresh plates . At the same time , unmated hermaphrodites that never encountered a male were also moved to fresh plates at the same density ( approximately 30 worms per plate ) . After approximately 24 hr , 40 worms from each condition ( individuals of each age and genotype that either received male sperm or never interacted with males ) were picked onto an unseeded NGM plate . These worms were immediately washed twice with ice cold M9 buffer ( 22 mM KH2PO4 , 42 mM Na2PO4 , 86 mM NaCl , and 1 mM MgSO4 ) and the worm pellets were flash frozen in liquid nitrogen . The remaining worms were used to measure their lifespan ( Figure 2A–D and Supplementary file 2 ) as described in the section ‘Lifespan Assays’ . RNA was extracted from the flash frozen worm pellets with 500 μL Trizol and 200 μL chloroform followed by 250 μL phenol and 200 μL chloroform extractions and finally , an isopropanol precipitation . Remaining DNA was degraded with DNaseI ( Promega ) and the RNA cleaned with a sodium acetate and ethanol precipitation . RNA quality was measured using Nanodrop spectrophotometry and the Agilent BioAnalyzer Total RNA Nano chip and kit . mRNA enriched cDNA was prepared using 10 ng of total RNA ( quantified by Nanodrop spectrophotometry ) and the Takara SMART-seq v4 Ultra Low Input RNA kit , with 8 rounds of amplification . Paired-end libraries were made using the Nextera XT DNA library prep kit ( Illumina ) with 1 ng of cDNA ( quantified using the Qubit dsDNA High Sensitivity reagents , Invitrogen ) and barcoded using the Nextera XT Index Kit v2 ( Illumina ) . Libraries were purified with 30 μL AMPure XP beads ( Beckman Coulter ) as directed in the Nextera XT kit . Library quality and quantity were assessed using the Agilent Bioanalyxer High Sensitivity DNA Assay . All samples and biological replicates ( A-D ) were pooled and sequenced on a single Illumina NextSeq run . Paired-end , 75 base pair sequencing was performed . RNA-seq reads were aligned to the WBcel235 genome and gene read counts were calculated using STAR ( version 2 . 5 . 4a ) . Low-coverage genes that had less than one read count per million mapped reads in less than three samples were filtered out . Data were normalized with a variance-stabilizing transformation ( DESeq2 version 1 . 10 . 1 ) prior to Principal Component Analysis ( PCA ) in R ( version 3 . 2 . 4 and Biobase version 2 . 30 . 0 ) . PCA was carried out using the R method ( prcomp ) . Differential expression was calculated using DESeq2 ( version 1 . 10 . 1 ) . The results from DESeq2 can be found in Figure 2—source data 1 . Gene Ontology ( GO ) enrichment was performed in R using the Fischer’s t-test and Benjamini-Hochberg corrected . Full GO enrichment results can be found in Figure 2—source data 2 . Heatmaps were generated in R using normalized read counts ( variance-stabilizing transformation ) . Code is available online ( https://github . com/brunetlab/Booth_etal_2019 . git ) . The CEH-18 network was determined using the GeneMANIA webserver ( Warde-Farley et al . , 2010 ) . The default settings were used with the exception of the types of interactions allowed ( predicted interactions and protein domain similarity were deselected ) . Networks were re-drawn based on the GeneMANIA output using Adobe Illustrator to incorporate the DESeq2 output from the RNA-seq ( Figure 2—source data 1 ) . P-values for enrichment of differentially expressed genes in the network were calculated using the hypergeometric distribution . The PSWM file for the CEH-18 DNA binding motif ( Narasimhan et al . , 2015 ) was downloaded from http://cisbp . ccbr . utoronto . ca and a publicly available CEH-18 ChIP-seq dataset ( Kudron et al . , 2018 ) was downloaded from http://epic . gs . washington . edu/modERN/ . The ChIP-seq peaks were then converted to FASTA format with bedtools ‘getfasta’ ( Quinlan and Hall , 2010 ) to be made compatible with MEME . The MEME Suite AME ( with default parameters ‘Average odds score’ and ‘Fisher’s exact test’ using shuffled input sequences as the control ) was used with the CEH-18 motif to determine statistical enrichment of the CEH-18 motif within the CEH-18 binding peaks ( McLeay and Bailey , 2010 ) . This analysis revealed significant enrichment for the CEH-18 DNA binding motif in the CEH-18 ChIP-seq peaks ( p=4 . 77×10−5 ) , though the enrichment was not strong . As a comparison , the same analysis was performed using the DAF-16 PSWM file ( http://cisbp . ccbr . utoronto . ca; Weirauch et al . , 2014 ) to test for enrichment within DAF-16 ChIP-seq peaks ( http://data . modencode . org; Contrino et al . , 2012 ) . There was very high enrichment of the DAF-16 DNA binding motif within DAF-16 ChIP-seq peaks ( p=1 . 44×10−359 ) . We next tested for the enrichment of the CEH-18 DNA binding motif in the sequences surrounding the transcription start sites of the differentially expressed genes from the RNA-seq . The ChIPSeeker ( Yu et al . , 2015 ) function ‘getPromoters’ was used with parameters ‘TxDb = TxDb . Celegans . UCSC . ce11 . refGene , upstream = 300 , downstream = 300’ to assign promoters to genes from lists of differentially expressed genes generated from the RNA-seq data with a statistical significance threshold of FDR < 0 . 05 . The resulting bed files were converted to FASTA format and inspected for motif enrichment as above using the MEME suite AME ( McLeay and Bailey , 2010; Quinlan and Hall , 2010 ) . To identify differentially expressed genes that have a nearby CEH-18 binding site , the publicly available CEH-18 ChIP-seq peaks ( Kudron et al . , 2018 ) were first de-duplicated to remove any non-unique lines , and the bed file was converted to a gRanges object using readBed ( ) from the ‘genomation’ package ( Akalin et al . , 2015 ) . The gRanges object was then annotated using annotatePeak ( ) from the ‘ChIPSeeker’ package ( Yu et al . , 2015 ) to associate peaks with a proximal gene . The CEH-18 ChIP-seq peaks that were associated with genes differentially regulated in the RNA-seq datasets were then outputted based on the annotated gene associations ( from ChIPSeeker; Yu et al . , 2015 ) . This output is Figure 4—source data 1 . Using the hypergeometric distribution test , we found that the differentially expressed genes from our RNA-seq ( comparing young hermaphrodites to young feminized individuals , mated and unmated ) were not significantly enriched for the CEH-18 binding sites . We note that our RNA-seq was performed using whole worms and that the differentially expressed genes from these data are the result of transcripts from all the tissues , many of which may not express ceh-18 . The lack of transcription factor binding enrichment , particularly in transcription factors acting in a cell- or tissue-specific manner , has been described in C . elegans ( Cao et al . , 2017; Narasimhan et al . , 2015 ) and is thought to be largely due to cell heterogeneity . Cell- or tissue-specific methods including single-cell RNA-seq could better elucidate the CEH-18 regulatory network in the somatic gonad . We then generated a list of differentially expressed genes between young hermaphrodites and young feminized individuals ( unmated and that mated with males ) that have a neighboring CEH-18 binding site by associating ChIP-seq binding peaks with nearest transcription start site using ChIPSeeker ( Yu et al . , 2015 ) . These data are in Figure 4—source data 1 . This subset of genes was then used for GO enrichment in R using the Fischer’s t-test and Benjamini-Hochberg correction . The output for the GO enrichment is Figure 4—source data 2 . As a negative control , we also performed this analysis with an unrelated transcription factor , MAB-5 and these data are presented in Figure 4—source data 1 and 2 . All code for these analyses are available online ( https://github . com/brunetlab/Booth_etal_2019 . git ) . To create a feminized C . briggsae worm that is competent for RNAi knock-down by ingested dsRNA , we crossed the she-1 ( v35 ) feminized strain to a transgenic strain ( mfIs42[Cel-sid-2; Cel-myo-2::DsRed] ) that is susceptible to ingested dsRNA ( Nuez and Félix , 2012 ) . Worms that were homozygous for the she-1 ( v35 ) mutation were determined by PCR followed by Sanger sequencing using the following primers: 5’- CAATTGTCATGCGACCAGATTT −3’ 5’- GCTTGTCCGAAACCAATGAAC −3’ The homozygous presence of the mfIs42[Cel-sid-2; Cel-myo-2::DsRed] integration was determined by observing transmission of the DsRed marker in all progeny for several generations . Portions of the highly conserved Cbr-ceh-18 and Cbr-vab-1 genes ( Figure 5—figure supplement 2A ) were amplified from C . briggsae ( strain AF16 ) genomic DNA and the DsRed transgenic marker ( from JU1018 genomic DNA ) using Platinum HiFi Supermix ( Invitrogen ) and the following primers: Cbr-ceh-18: 5’-GGTCCTCGAGGTATTCACCAACGGCAACAAC-3’ and 5’-GCGTACTAGTGGTCCTCTTCCTTCTTCTCTTG-3’ Cbr-vab-1: 5’-GGTCCTCGAGAGTGTGGATCCGTTGTGATG-3’ and 5’-GCGTACTAGTGGAAATCCAACTCACCCTATGA-3’ dsRed: 5’-GGTCCTCGAGGAACGTCATCACCGAGTTCAT-3’ and 5’-GCGTACTAGTGATGGTGTAGTCCTCGTTGTG-3’ The PCR products and the L4440 vector were digested with SpeI and XhoI ( New England Biolabs ) . Ligation was performed with T4 ligase ( New England Biolabs ) . The ligation products were first transformed into TOP10 chemically competent cells ( Invitrogen ) . After verification of the plasmids by Sanger sequencing , the correct plasmids were transformed into chemically competent HT115 E . coli . HT115 bacteria containing these plasmids were used for RNAi knock-down by feeding in C . briggsae ( mfIs42[Cel-sid-2; Cel-myo-2::DsRed] ) ( see ‘Lifespan Assays’ above ) . Worms were prepared for imaging using 1 mM sodium azide and mounted on a 2% agarose pad . The images presented and quantified in Figure 5—figure supplement 3 were taken using a Zeiss Axioskop 2 Plus . All images for an experiment were taken using the same exposure length . DsRed fluorescence was quantified in Fiji ( Schindelin et al . , 2012 ) using the mean gray value of the pharynx bulb . To measure the conservation of orthologs of the sperm-sensing proteins CEH-18 and VAB-1 , the sequences of these proteins were downloaded from www . wormbase . org ( WBcel235 ) and aligned using MUSCLE v3 . 8 ( Edgar , 2004a; Edgar , 2004b ) . These protein alignments were input to the JalView visualization tool ( Waterhouse et al . , 2009 ) to generate Figure 5—figure supplement 2A . Protein domains ( pfam ) were determined by www . wormbase . org . Data are available in the main text or supplementary materials . RNA-seq reads are available online at NCBI SRA ( PRJNA508378 ) and all code used for RNA-seq analysis is available online ( https://github . com/brunetlab/Booth_etal_2019 . git; copy archived at https://github . com/elifesciences-publications/Booth_etal_2019; Booth , 2019 ) .
Sexual interactions have a potent influence on health in several species , including mammals . Previous work in C . elegans identified strategies used by males to accelerate the demise of the opposite sex ( hermaphrodites ) . But whether hermaphrodites evolved counter-strategies against males remains unknown . Here we discover that young C . elegans hermaphrodites are remarkably resistant to brief sexual encounters with males , whereas older hermaphrodites succumb prematurely . Surprisingly , it is not their youthfulness that protects young hermaphrodites , but the fact that they have self-sperm . The beneficial effect of self-sperm is mediated by a sperm-sensing pathway acting on the soma rather than by fertilization . Activation of this pathway in females triggers protection from the negative impact of males . Interestingly , the role of self-sperm in protecting against the detrimental effects of males evolved independently in hermaphroditic nematodes . Endogenous strategies to delay the negative effect of mating may represent a key evolutionary innovation to maximize reproductive success .
A nematode worm known as Caenorhabditis elegans is often used in the laboratory to study how animals grow and develop . There are two types of C . elegans worm: hermaphrodite individuals produce both female sex cells ( eggs ) and male sex cells ( sperm ) , while male individuals only produce sperm . The hermaphrodite worms are able to reproduce without mating with another worm , allowing populations of C . elegans to grow rapidly when they are living in favorable conditions . However , when the hermaphrodites do mate with males they tend to produce more offspring . These offspring are also usually healthier because they receive a mixture of genetic material from two different parents . Although mating is beneficial for the survival of a species it can also harm an individual animal . Previous studies have shown that mating with male worms can accelerate aging of hermaphrodite worms and cause premature death . However , it remained unclear whether hermaphrodite worms have evolved any mechanisms to protect themselves after mating with a male . To address this question , Booth et al . used genetic techniques to study the lifespans of hermaphrodite worms . The experiments found that the hermaphrodites’ own sperm ( known as self-sperm ) regulated a sperm-sensing signaling pathway that protected them from the negative impact of mating with males . Hermaphrodites with self-sperm that mated with males lived for a similar length of time as hermaphrodites that did not mate . On the other hand , hermaphrodites that did not have self-sperm ( because they were older or had a genetic mutation ) had shorter lifespans after mating than worms that did not mate . Modulating the sperm-sensing signaling pathway in worms that lacked self-sperm was sufficient to protect them from the negative effects of mating with males . Further experiments found that the hermaphrodites of another nematode worm called C . briggsae – which evolved self-sperm independently of C . elegans – also protected themselves from the negative effects of mating with males in a similar way . This suggests that other animals may also have evolved similar mechanisms to protect themselves from harm when mating . A separate study by Shi et al . has found that the beneficial effects of self-sperm are mediated by a pathway linked to longevity that also exists in mammals . The results of both investigations combined suggest possible avenues for future research into the complex relationship between health , longevity , and reproduction .