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Vol. 16, Issue 9, 3978-3986, September 2005

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Inositol 1,4,5-Trisphosphate Signaling Regulates Mating Behavior in Caenorhabditis elegans Males

Nicholas J. D. Gower ^*  , Denise S. Walker ^* , and Howard A. Baylis ^*

^* Department of Zoology, University of Cambridge, Cambridge CB2 3EJ, United Kingdom; Laboratory of Receptor Signaling, The Babraham Institute, Cambridge CB2 4AT, United Kingdom

Submitted February 4, 2005; Revised May 11, 2005; Accepted June 3, 2005
Monitoring Editor: Martin Chalfie

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Complex behavior requires the coordinated action of the nervous system and nonneuronal targets. Male mating in Caenorhabditis elegans consists of a series of defined behavioral steps that lead to the physiological outcomes required for successful impregnation. We demonstrate that signaling mediated by inositol 1,4,5-trisphosphate (IP³) is required at several points during mating. Disruption of IP₃ receptor (itr-1) function results in dramatic loss of male fertility, due to defects in turning behavior (during vulva location), spicule insertion and sperm transfer. To elucidate the signaling pathways responsible, we knocked down the six C. elegans genes encoding phospholipase C (PLC) family members. egl-8, which encodes PLC-, functions in spicule insertion and sperm transfer. itr-1 and egl-8 are widely expressed in the male reproductive system. An itr-1 gain-of-function mutation rescues infertility caused by egl-8 RNA interference, indicating that egl-8 and itr-1 function together as central components of the signaling events controlling sperm transfer.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Complex behaviors in animals require not only processing and production of information by the nervous system, but also coordinated changes in the physiology of the target organs required for the execution of that behavior. Male mating behavior in Caenorhabditis elegans is an example. Male mating relies on specialized sex-specific structures and is one of the most complex behavioral and physiological processes exhibited by C. elegans. Mating begins with the posterior of the male making contact with the hermaphrodite; the male places his tail against the hermaphrodite and moves along its body. When the head or tail of the hermaphrodite is reached a turn is made via a deep ventral flexion, this behavior continues until the vulva is located. The spicules, a pair of copulatory structures, are then inserted into the vulva and sperm is transferred through the vas deferens, to the hermaphrodite uterus (Sulston et al., 1980; Loer and Kenyon, 1993; Liu and Sternberg, 1995). This behavior thus requires integration of sensory information, coordinated body movements, and coordinated muscle activity to bring about spicule insertion and the correctly timed release of sperm.

The sequence of mating activities that culminates in sperm transfer requires many levels of regulation, and their coordination is clearly based on many inter- and intracellular signaling events. The second messenger inositol 1,4,5-trisphosphate (IP₃) is a prime candidate for playing a signaling role at the interface of neuronal and target organ physiology. IP₃ signaling transduces signals from a wide range of extracellular stimuli into intracellular Ca²⁺ signals that regulate cell physiology. IP₃, produced by the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) by activated PLC, diffuses through the cytosol and causes Ca²⁺ release from intracellular stores through the activation of IP₃Rs (Berridge, 1993).

IP₃Rs in C. elegans are encoded by a single gene, itr-1 and are widely expressed throughout the animal (Baylis et al., 1999; Dal Santo et al., 1999; Gower et al., 2001). Genetic and transgenic approaches in C. elegans have led to the characterization of roles for IP₃ signaling. A genetic screen for genes that act downstream of let-23 (epidermal growth factor [EGF] receptor) identified itr-1 as a component of a signaling pathway regulating ovulation (Clandinin et al., 1998). Part of this requirement stems from the importance of IP₃ signaling in the regulation of gonadal sheath cell contraction (Yin et al., 2004). Analysis of itr-1 loss-of-function mutants led to the identification of a central role in the regulation of the defecation motor program (Dal Santo et al., 1999). We have demonstrated, using itr-1 mutants, RNA-mediated interference (RNAi) and an inducible dominant-negative construct (IP₃ sponge), that IP₃R and IP₃ signaling are involved in the up-regulation of pharyngeal pumping in response to food and in multiple stages of embryogenesis (Walker et al., 2002).

Ca²⁺ release is a key integratory intracellular signaling event and is central to a wide range of cellular responses (Berridge, 1993, 1997; Clapham, 1995). The spatial and temporal structure of Ca²⁺ signals is regulated through components of the Ca²⁺ signaling toolkit (Berridge et al., 2000). Previous studies have identified components of the Ca²⁺ signaling network in the regulation of male mating behavior. The unc-68-encoded ryanodine receptor sarcoplasmic reticulum Ca²⁺ channel and the egl-19-encoded L-type voltage-gated Ca²⁺ channel ₁ subunit play a role in spicule insertion through their action in protractor muscles (Garcia et al., 2001; Maryon et al., 1998).

To further dissect the Ca²⁺ signaling networks that modulate this complex series of behaviors, we have determined the role of IP₃-mediated signaling in male mating behavior. We describe results that implicate IP₃R-mediated Ca²⁺ release as being important for correct execution of turning and spicule insertion. We demonstrate that IP₃ signaling is fundamental to the process of sperm transfer in C. elegans males and provide evidence that EGL-8 (PLC-) is the specific PLC that functions in the generation of this IP₃ signal.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
C. elegans Strains and Culture
C. elegans were maintained using standard culture methods (Lewis and Fleming, 1995). The following strains were used: Bristol N2 wild-type; JT73 (itr-1 (sa73) IV, Iwasaki et al., 1995); CB1091 (unc-13 (e1091) I, Waterston and Brenner, 1978); BA1 (fer-1 (hc1) I, Ward and Miwa, 1978); CB1467 (him-5 (e1467) V, Hodgkin et al., 1979); CB1489 (him-8 (e1489) IV, Hodgkin et al., 1979); and CB138 (unc-24 (e138) IV, Brenner, 1974). HB100 (itr-1 (sa73); him-5 (e1467)) was constructed by mating CB1467 males and JT73 hermaphrodites. HB101 and HB102 (itr-1 (sy290) unc-24 (e138) IV; him-5 (e1467) V jwEx101 [unc-24 (+), mec-7::gfp]) were constructed by first crossing PS2582 hermaphrodites with CB1467 males and then rescuing unc-24 (e138) by transgenic expression of wild-type unc-24. A genomic DNA fragment from 1 kb upstream of the unc-24 start codon to 600 base pairs downstream (bp 11573–15174 of cosmid F57H12) was amplified by PCR and ligated into pGEM-T (Promega, Madison, WI) to give pNG135. PS2582 was injected with pNG135 and pPD117.01 (a mec-7::gfp reporter construct that expresses in mechanosensory neurons) as a marker. HB103 (unc-24 (e138) IV; him-5 (e1467) V jwEx101 [unc-24 (+), mec-7::gfp]) was constructed in a similar way, except that CB138 was used instead of PS2582. The resulting strains display normal movement.

RNA-mediated Interference
RNAi of itr-1 was carried out using Escherichia coli HT115 carrying derivatives of the vector pPD129.36 (Timmons et al., 2001), which contains two flanking T7 RNA polymerase promoters. For RNAi of itr-1 we used pNG030 (Walker et al., 2002), which carries a 1-kb region of the itr-1 cDNA. For RNAi of PLC genes we constructed derivatives of pPD129.36, carrying 1 kb of the cDNA of each PLC gene; these are pHAB301 (egl-8), pHAB302 (plc-1) pHAB303 (plc-3) pHAB304 (plc-4), pHAB305, (plc-2), and pHAB306 (pll-1). As a control we used pPD128.110, a derivative of pPD129.36 with a gfp cDNA insert (Timmons et al., 2001). Five L4 him-8 (e1489) hermaphrodites were placed onto each feeding plate and incubated at 25°C for 24 h and then transferred onto separate feeding plates and allowed to lay eggs for 24 h at 25°C. L4 male offspring were then removed for functional analysis.

Fertility Assays
To assay male fertility we used fer-1 (hc1) "females" (Ward and Miwa, 1978) as recipients. Both 1:1 and 12:4 male to female crosses were set up and scored in the same way: 30-mm NGM plates (2.5% agar, to prevent burrowing) were poured, seeded the following day with a 5-mm diameter E. coli lawn, and used for fertility assays the following day. Fer-1 (hc1) animals were maintained at the permissive temperature of 15°C, at which they are fertile. Adult hermaphrodites were then transferred to the restrictive temperature of 25°C and allowed to lay eggs for 24 h, and the offspring were allowed to develop at 25°C. Either 1 or 4 L4 larvae were then transferred to the E. coli spot on the cross plate, with either 1 or 12 RNAi-treated or mutant males. Crosses were performed at 25°C and offspring were counted up to a maximum of 100. Fertility was compared with fer-1 (hc1) only (i.e., no males) plates. To remove the background fertility we subtracted the highest fer-1 (hc1) only control plate value from the number of progeny on each test plate. A plate was deemed to be fertile if the number of offspring exceeded the highest number observed on a fer-1 (hc1) only plate.

Scoring Male Behaviors
Male mating behaviors were assayed by placing a single young adult male at t = 0 in the center of a plate containing 30, evenly distributed, young adult unc-13 (e1091) hermaphrodites. Unc-13 mutant hermaphrodites were used because greater mating efficiencies are observed when the hermaphrodites are unable to move (Hodgkin, 1983). To control for variation of mating activity due to age of plate (Liu and Sternberg, 1995), 30-mm agar plates were poured one day, seeded with a 5-mm lawn of E. coli OP50 the next day, and used for mating behavior assays the following day. Male mating behavior (Loer and Kenyon, 1993; Liu and Sternberg, 1995) was assessed during a 20-min observation period using the following criteria: hermaphrodite body contact, backing with tail contacting the hermaphrodite, turning of the tail around the circumference or an end of the hermaphrodite, forward movement of tail in contact with hermaphrodite, general vulva location movements, fine tuning vulva location movements, attachment of the tail to the vulva, spicule prodding, and spicule insertion. All observations of the cross plates were made at 400x on a Zeiss Axiovert S100 inverted microscope (Thornwood, NY; 23–25°C).

To score turning ability crosses were set up as described above but only turns were scored in the 20-min period. A turn or attempted turn was scored when the male backed up to the end (head or tail) of a hermaphrodite and attempted to curl his tail to contact the opposite surface of the mate. Each such turn was categorized (Loer and Kenyon, 1993) as follows: "good" (tail remained in contact with the hermaphrodite throughout the turn and continued backing on the opposite side after the turn was complete), "sloppy" (tail temporarily lost contact, but quickly regained contact on the opposite side because of a favorable trajectory), or "missed" (tail sailed off the end of the hermaphrodite, completely losing contact).

Construction of Transgenic Animals Carrying GFP Fusions and Constructs
Three itr-1::gfp constructs had previously been constructed (Gower et al., 2001); pNG007 contains the 650-base pair sequence upstream of exon 1; pGT001 contains the 2.4-kb sequence that separates exon 1 and exon 2, and pGT002 contains the 4.4-kb sequence that separates exon 2 and the first common exon, 4. plc::gfp fusions were made using a PCR fusion-based approach (Hobert, 2002). Genomic fragments were amplified for each member of the family; egl-8 was amplified from 4 kb upstream to exon 6; plc-1 was amplified from 4 kb upstream to exon 2; plc-2 was amplified from 1.8 kb upstream to exon 14; plc-3 was amplified 3.4 kb upstream to exon 8; plc-4 was amplified from 4.2 kb upstream to just upstream of the start site; and pll-1 was amplified from 4 kb upstream to exon 3. PCR products were the fused in-frame to the gfp gene. DNA for microinjection was prepared using Qiaprep Spin Miniprep kit (Qiagen, Valencia, CA) and further purified by precipitation with ethanol in the presence of 0.1 M potassium acetate, before micro-injection into him-8 (e1489) hermaphrodites (Hodgkin et al., 1979; Mello and Fire, 1995). Images were recorded using a Leica SP confocal microscope (Bannockburn, IL). Images are maximum projections of a Z-series.

Fluorescent Sperm Tracking
Sperm tracking assays used the method of Hill and L'Hernault (2001). L4 virgin males were picked from the RNAi feeding plates to a freshly seeded plate and grown overnight in the absence of hermaphrodites. Males were incubated in 70 µM SYTO17 dye (Molecular Probes, Eugene, OR) at 25°C for 3 h, transferred to an NGM plate and allowed to recover, and then picked for mating experiments. Twelve males and 4 fer-1 (hc1) females were picked onto a cross plate and allowed to mate at 25°C for 24 h. Recipient females were mounted onto 2% agar pads and examined by differential interference contrast (DIC) and fluorescence microscopy to locate male-derived (fluorescent) sperm. The level of sperm transferred was classified as good (>10 sperm visible in recipient), weak (between 1 and 10 sperm visible), and none.

Small Scale Sperm Isolation
Three males were placed in a 7-µl drop of SM (L'Hernault and Roberts, 1995) containing 1 mg/ml bovine serum albumin and 200 µg/ml pronase. Sperm were then released by cutting just in front of the tail with a razor blade (L'Hernault and Roberts, 1995). Spermatids were released into the solution and 80–95% became activated to form spermatozoa within 5 min. Sperm were examined using DIC optics on a Zeiss Axiovert S100 inverted microscope.

Spicule Protraction
Spicule protraction in response to levamisole was assayed as described by Garcia et al. (2001). Twenty to 40 animals were tested for each concentration. EC₅₀ values were calculated by fitting sigmoidal curves to log-transformed data using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA).

Statistical Analysis
All statistical analysis was carried out using Chi-squared tests.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
itr-1 Function Is Essential for C. elegans Male Fertility
The ability of males to mate successfully is dependent on functions endowed by a range of male-specific structures. Therefore defects in the function or development of any of these structures will result in reduced male fertility. To test whether itr-1 is required for fertility, we performed mating assays between itr-1 RNAi males and fer-1 (hc1) "females" (Ward and Miwa, 1978). We performed RNAi on him-8 (e1489) (Hodgkin et al., 1979), which produces a high incidence of males. Fer-1 (hc1) is a temperature-sensitive allele that results in sterility at 25°C, due to an inability to produce active sperm (Ward and Miwa, 1978). At the restrictive temperature, fer-1 (hc1) animals therefore require the contribution of functional sperm from males for successful fertilization. We performed crosses between single fer-1 (hc1) hermaphrodites and test males, i.e., 1:1 crosses. In this situation, even in the presence of wild-type males, not all plates yield progeny. These assays clearly demonstrated that males in which itr-1 is knocked down using RNAi have drastically reduced fertility when compared with control males (Figure 1A). Because fer-1 (hc1) animals have a low level of background fertility, this was subtracted from the number of offspring produced in each cross, before calculating the percentage of fertile crosses (see Materials and Methods). Using this assay, 52% of control crosses were fertile compared with only 4% of crosses using itr-1 RNAi males.

View larger version (27K): [in this window] [in a new window]   Figure 1. itr-1 functions in male fertility. (A) Fifty 1:1 crosses between fer-1 (hc1) "females," which are unable to produce active sperm and therefore require male-derived sperm for fertilization to occur, and males generated by feeding dsRNA specific for gfp (i) or itr-1 (ii), compared with fer-1 (hc1) only mock crosses (iii). For simplicity, crosses that produced 100 or more offspring are classed as having produced 100. (B) The role of itr-1 in male mating behavior. A single male was placed at the center of a seeded NGM plate with 30 unc-13 (e1091) hermaphrodites and observed for 20 min. Graph shows the percentage of males that performed the behavior at least once during the observation period. Performance of a given step implies successful performance of preceding steps; n = 17 (gfp RNAi) and 16 (itr-1 RNAi). (C) Turning ability of itr-1 and gfp RNAi males was determined by observing males as in B, but for 10 min. Turns were classified as missed, sloppy, or good (see Materials and Methods); n = 23 for each treatment.

itr-1 Functions in Male Mating Behavior
The mating behavior of males follows a set sequence of steps culminating in sperm transfer (Loer and Kenyon, 1993; Liu and Sternberg, 1995). The key stages are; hermaphrodite recognition, backing, turning, vulval location, and copulation. Each of these steps can be further divided into subcategories. In particular, copulation is divided into spicule prodding (i.e., repeated contraction and relaxation of spicule protractor muscles), spicule insertion (i.e., prolonged spicule protractor contraction), and sperm transfer. To determine whether reduction in itr-1 function disrupted any of these steps, we scored him-8 (e1489) itr-1 RNAi animals for their ability to execute each of these behaviors properly (Figure 1B). itr-1 RNAi males responded to the hermaphrodites by backing and performed turning, backing, and vulval location with a frequency similar to that of the control males. itr-1 RNAi males showed a very slight reduction in spicule prodding, whereas spicule insertion occurred at <25% of the frequency observed for control males (p < 0.05).

Although itr-1 RNAi males performed turns with a similar frequency to control animals, these turns were often poorly executed. A normal turn consists of a sharp ventral arch of the tail, performed at the proper time (Liu and Sternberg, 1995). Loer and Kenyon (1993) have produced a classification for turns, in which they are deemed to be "good," "sloppy," or "missed" (see Materials and Methods). itr-1 RNAi males (Figure 1C) perform significantly fewer good turns than the control males (p < 0.001), the number of sloppy turns is substantially increased, and the number of missed turns is also increased. Thus itr-1 males fail to coordinate turns properly. These results demonstrate that ITR-1 has a role in regulating male turning and prolonged spicule insertion. Nevertheless, these results do not explain the extent of sterility observed, as discussed later.

itr-1 Is Expressed in Many Male-specific Structures
To identify cells and tissues that might correlate with the functional requirements for itr-1, we determined the expression pattern of itr-1 in males. The expression pattern of itr-1 has been well characterized in hermaphrodites using transgenic animals carrying itr-1::gfp reporter constructs and antibody staining (Baylis et al., 1999; Dal Santo et al., 1999; Gower et al., 2001). The itr-1 gene contains three promoters, each of which drives tissue-specific expression of an alternative itr-1 mRNA (Gower et al., 2001). Therefore, we made transgenic him-8 (e1489) lines carrying itr-1::gfp reporter constructs for each promoter and determined their expression pattern in males.

Expression in male-specific structures is directed by all three promoters. Promoter pA directs expression in the spicule protractor muscles of the proctodeum and in a single male-specific neuron that is likely to be CP8 or CP9 (Figure 2A). The spicule protractor muscles are required for the prodding of the spicule before vulval insertion and for the maintenance of spicule insertion while sperm is transferred (Garcia et al., 2001). Promoter pB directs expression in the spicule retractor muscles, gubernaculum retractor muscles, posterior oblique muscles, diagonal muscles, and the vas deferens (Figure 2B). The spicule retractors and gubernaculum retractors are required for spicule retraction after sperm transfer (Garcia et al., 2001). The posterior oblique muscles and diagonal muscles contribute to the ventral flexure of the male tail and ablation of the diagonal muscles results in animals that never make normal turns (Loer and Kenyon, 1993). The vas deferens connects the seminal vesicle to the proctoderm and is involved in sperm transfer (Emmons and Sternberg, 1997). Promoter pC also directs expression in the vas deferens, as well as the seminal vesicle and the valve that separates the seminal vesicle and vas deferens (Figure 2C). Thus itr-1 is expressed widely in the sex-associated muscles and somatic gonad of males.

View larger version (68K): [in this window] [in a new window]   Figure 2. itr-1 is expressed in male-specific structures. (A) Schematic diagram showing the male-specific structures discussed in this report. Spic Retr, spicule retractor muscles; Spic Pro, spicule protractor muscles; Gub Retr, gubernaculum retractor; Ant Ob, anterior oblique; Gub Erec, gubernaculum erector; PostOb, posterior oblique; Vas def, vas deferens; Diag, diagonal muscles. GFP reporter analysis of the three itr-1 promoters. (B) Promoter pA directs expression in the spicule protractor muscles and in a male-specific neuron. (C) Promoter pB directs expression in the vas deferens, diagonal muscles, gubernaculum retractor, posterior oblique muscles, and spicule retractor muscles. (D) Promoter pC directs expression in the vas deferens, valve cell, and seminal vesicle (SEM Ves).

A live fluorescent sperm-tracking assay, to assess the insemination, activation, and spermathecal targeting of male-derived sperm, has previously been described (Hill and L'Hernault, 2001). him-8 (e1489) itr-1 RNAi animals and itr-1 (sa73) him-5 (e1467) males were treated with the fluorescent dye SYTO 17 (Figure 3A; see Materials and Methods) to label their sperm and were then used in 12:4 fertility assays as described previously. Knockdown of itr-1 resulted in a significant (p < 0.001) reduction in sperm transfer, with the majority of 12:4 crosses showing a complete lack of transfer. Similarly, itr-1 (sa73) males showed a significant (p < 0.001) decrease in the efficiency of sperm transfer, compared with controls (Figure 3B). This sperm transfer defect goes further to explain the fertility defect observed and is sufficient to account for the loss of fertility observed.

View larger version (69K): [in this window] [in a new window]   Figure 3. itr-1 mutant males are defective in sperm transfer, but produce normal sperm. Males were stained with SYTO 17 (Ai) and mated with fer-1 (hc1) "females" in a 12:4 ratio. Twenty-four hours later recipient "females" were analyzed for labeled sperm in their spermatheca (Aii, arrow). Levels of transferred sperm were classified as none, weak, or good (see Materials and Methods); n = 40. (B) After pronase treatment, isolated itr-1 RNAi male-derived spermatids develop (C, i–iv) in the same way as gfp RNAi male-derived spermatids (D, i–iv).

Phospholipase-C Functions in Male Fertility
IP₃ production is triggered by the rapid hydrolysis of PIP₂ by PLC isozymes. In C. elegans six PLC-like genes have been identified in the genome (see Supplementary Data). They correspond to vertebrate PLC- (Lackner et al., 1999; Miller et al., 1999), PLC-, PLC- (Yin et al., 2004), PLC-, PLC-like (Shibatohge et al., 1998), and an unusual protein that is most like PLC- and is referred to as -like. To determine which PLCs function upstream of the IP₃R in the regulation of male-specific behaviors, we used RNAi to assay their contribution to male fertility. Using RNAi allowed us to test all six genes (most have no known mutants) and to avoid the locomotion defects associated with egl-8 (PLC-) mutants. Males treated with gene-specific dsRNA were tested in the 12:4 mating assay for fertility (Table 1). egl-8 RNAi males showed a dramatic loss of fertility, whereas none of the remaining five PLC gene knockdowns produced fertility defects. This implicates egl-8 as a possible upstream component of one or more of the mating functions that have been identified for itr-1.

Members of the plc Gene Family Are Expressed in Male-specific Structures
The expression pattern of the six PLC genes was determined in males by establishing transgenic him-8 (e1489) animals carrying green fluorescent protein (GFP) reporter fusions. The fusions contained up to 4-kb genomic sequence up-stream of the start site and gfp was fused in frame to the PLC gene (see Materials and Methods for details). egl-8 is expressed in the vas deferens, spicule protractor muscles, diagonal muscles and a male-specific neuron that is probably CP8 or CP9 (Figure 4A, i and ii) and is also widely expressed in the nervous system, as expected from previous work (Lackner et al., 1999; Miller et al., 1999). plc-3 (encoding PLC-) is expressed in the vas deferens and in the valve cell that separates the seminal vesicle and vas deferens (Figure 4Aiii). plc-4 (encoding PLC-) is expressed in the vas deferens (Figure 4Aiv). GFP reporters for plc-1, plc-2, and pll-1 are not expressed in male-specific structures. Thus three different PLC subtypes are expressed in the somatic gonad of the male, suggesting that complex signaling pathways may exist in the vas deferens; however, in the context of male fertility it seems that only egl-8 is essential.

View larger version (45K): [in this window] [in a new window]   Figure 4. The roles of C. elegans phospholipase C genes in male fertility. (A) Expression patterns of the phospholipase C family in males were determined using GFP reporter constructs. egl-8::gfp (A, i and ii) is expressed in the diagonal muscles, spicule retractors, and vas deferens and a male-specific neuron; plc-3::gfp (Aiii), is expressed in the valve cell and in the vas deferens, and plc-4::gfp (Aiv) is expressed in the vas deferens. plc-1, plc-2, and pll-1 reporters were not expressed in male-specific structures. (B) The role of egl-8 in male mating behavior was assessed as for Figure 1B; n = 20 for each treatment. (C) Turning ability of egl-8 RNAi males was scored as for Figure 1C.

To identify the fertility defect associated with egl-8 RNAi males, we performed behavioral assays on them (Figure 4B). Contact, turning, backing, vulva location, and spicule prodding were all performed at levels similar to that of the control animals. The percentage of animals that successfully inserted their spicules during the timed period was, however, reduced from 35 to 15% (although this is not statistically significant, p > 0.05). This level of reduction is similar to that observed on itr-1 RNAi, although the absolute levels for the control were lower in the egl-8 experiments, presumably reflecting environmental variability. This indicates that egl-8 may function upstream of the IP₃ signal, which we have shown to be involved in regulating spicule insertion.

Given that egl-8 is the only plc expressed in the diagonal muscles and that itr-1 seems to play a role in male turning, we scored egl-8 knockdown males for their turning ability (Figure 4C). We found that egl-8 knockdown males are capable of performing correct turns at the same frequency as the controls (p > 0.05). However, because egl-8 expression may not be effectively knocked down in these muscles and the nervous system that controls them, it is not possible to discount egl-8 from a function in turning.

itr-1 and egl-8 Function in Levamisole-induced Spicule Protraction
Spicule protraction is dependent on acetylcholine (ACh)-mediated signaling, and egl-30 (encoding G_q, which would be predicted to act upstream of egl-8 and itr-1) is also required for this process (Garcia et al., 2001). We therefore tested whether, like egl-30, itr-1 and egl-8 are required for the induction of spicule protraction by the nicotinic ACh receptor agonist levamisole. As Table 2 shows, knockdown of itr-1 results in a very substantial increase in the EC₅₀ of levamisole, whereas knockdown of egl-8 results in a more subtle, but nevertheless more than sixfold increase in EC₅₀. Thus both itr-1 and egl-8 function downstream of nicotinic ACh receptors in spicule protraction.

egl-8 RNAi Males Produce Active Sperm but Fail to Transfer Sperm to Hermaphrodites
As for itr-1, the observed reduction in mating efficiency does not explain the almost complete sterility of egl-8 RNAi animals so we investigated the effect on sperm transfer (Figure 5A). egl-8 RNAi males were also defective for sperm transfer (p < 0.001); of 31 recipient hermaphrodites tested only 1 contained a single transferred sperm. We found that egl-8 RNAi males produced normal numbers of spermatids, which were capable of undergoing spermiogenesis upon pronase treatment (Figure 5B, i–iii) in a manner that was identical to that observed for control RNAi males (Figure 5C, i–iii). These results demonstrate that egl-8 RNAi animals produce apparently normal sperm, and in support of this, both egl-8 mutant and egl-8 RNAi hermaphrodites produce fertilized eggs, demonstrating that hermaphrodites at least produce active sperm. Thus knock down of either egl-8 or itr-1 results in almost complete loss of sperm transfer, and furthermore, both genes are expressed in the vas deferens. These results implicate egl-8 as an upstream component in the itr-1-mediated pathway that regulates sperm transfer, and it seems plausible that the vas deferens is the site of function.

View larger version (68K): [in this window] [in a new window]   Figure 5. egl-8 mutant males are defective in sperm transfer, but produce wild-type sperm. (A) After SYTO 17 labeling, egl-8 RNAi males and control RNAi males were assessed for sperm transfer as for Figure 3B; n = 31. (B and C) Sperm were isolated from gfp and itr-1 RNAi males and activated in vitro by treatment with pronase. itr-1 RNAi male-derived spermatids developed (B, i–iii) in the same way as gfp RNAi male-derived spermatid (C, i–iii).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
itr-1 and egl-8 Function in Male Fertility
Male mating is a complex behavior comprising a series of steps that require correct processing of information by the nervous system and appropriate changes in physiology of the target organs. We have shown that the IP₃R is essential for the coordination of these events in C. elegans and that in the absence of IP₃R function males are sterile. We have demonstrated that the IP₃R functions in distinct events during male mating, each of which are required to varying degrees to enable a male to mate in the correct manner. Knockdown of egl-8, that encodes the C. elegans PLC-, also results in male sterility, which can be partially rescued by a gain of function mutation in itr-1. This study demonstrates the versatility of signaling through the IP₃R and reveals insight into how complex behaviors such as male mating are regulated.

egl-8 and itr-1 Function in Spicule Insertion
Spicule insertion occurs once the cloaca makes contact with the hermaphrodite vulva. Initially the spicule protractors contract and relax repeatedly, causing the spicules to prod the vulva. When the spicules penetrate the vulva, the protractors shorten while the retractor muscles lengthen, allowing the spicules to extend fully into the vulva (Garcia et al., 2001). Knockdown of either itr-1 or egl-8 resulted in a substantial reduction in spicule insertion. itr-1 is expressed in the spicule protractor and retractor muscles, whereas egl-8 seems only to be expressed in the protractors.

Garcia et al. (2001) have shown that the PCB, PCC, and SPC neurons induce protraction through the secretion of ACh. ACh receptors transduce this signal to either the ryanodine receptor (UNC-68), to promote prodding via periodic protractor muscle contraction through intracellular Ca²⁺ release, or to EGL-19, an L-type voltage-gated Ca²⁺ channel ₁ subunit, to promote prolonged protractor muscle contractions through Ca²⁺ influx. EGL-30, the C. elegans G_q protein, is implicated in the signal pathway downstream of the ACh response in the protractor muscles. ACh released from the PCB, PCC, and SPC sensory neurons induces muscle contraction through the activation of a number of ACh receptors. Garcia et al. (2001) have shown that agonists for both muscarinic and nicotinic ACh receptors activate multiple pathways in the protractor muscles and that mutations in egl-30 alter the muscle response to these agonists. These pathways differentially regulate unc-68 and egl-19 function and hence alter spicule prodding and prolonged spicule protraction differentially. EGL-30, like its vertebrate homologues, can stimulate PLC activity (Brundage et al., 1996), and egl-8 (PLC-) is known to function downstream of egl-30 in other muscle types (Lackner et al., 1999; Bastiani et al., 2003). It therefore seems likely that ITR-1 is involved in the Ca²⁺ signals required for protractor muscle contraction and that EGL-8, by catalyzing the production of IP₃, functions to couple EGL-30 activation with this Ca²⁺ release. This is supported by our observation that, like egl-30-deficient animals (Garcia et al., 2001), animals in which itr-1 or egl-8 have been knocked down are defective in spicule protraction induced by the nicotinic ACh receptor agonist levamisole.

The expression of itr-1 in the gubernaculum retractors suggests that itr-1 could also function in the initial movement of the spicules. Before insertion, the gubernaculum is thought to be responsible for moving the spicules to a more transverse position (Sulston et al., 1980). Therefore even if the spicule protractors are capable of inserting the spicules, they may be unable to if the spicules have not have been positioned correctly by the gubernaculum. Thus itr-1 may play multiple roles in spicule insertion; however, in this case activation of the IP₃R may not be mediated by egl-8, because it does not appear to be expressed in the gubernaculum.

EGL-8 Regulates Sperm Transfer through Activation of IP₃Rs
We have demonstrated that disruption of either itr-1 or egl-8 results in a dramatic defect in sperm transfer. Because the defects in mating behavior observed would be expected to have only very subtle effects on the results of 12:4 crosses, and because the spermatids produced appear to be normal, the defect in sperm transfer is likely to be the dominant factor contributing to the infertility observed. We have also demonstrated that an itr-1 mutation that increases affinity for IP₃ can partially rescue the sterility effect of knocking down egl-8 expression. This suggests that EGL-8 (PLC-), which is predicted to catalyze the production of IP₃, functions upstream of the IP₃R in the control of sperm transfer. Although there is evidence that diacyl glycerol can act as an effector for EGL-8 (Lackner et al., 1999) and that ITR-1 can act downstream of PLC-3 (Yin et al., 2004), this is the first demonstration that ITR-1 can also act as an effector for EGL-8.

We propose that the site of action for this IP₃-mediated pathway is the smooth muscles of the somatic gonad. More precisely, expression data for both itr-1 and egl-8 indicate that the sites of function are the vas deferens, seminal vesicle, and the valve that separates the two. The vas deferens has not been fully characterized but it is a complex structure of 30 cells, many of which appear to be secretory. After spicule insertion, in which the SPC motor neurons function by inducing the switch from periodic to prolonged spicule protraction (Garcia et al., 2001), a signal originating from the hermaphrodite uterus is required to induce the valve between the vas deferens and seminal vesicle to open, releasing spermatids (reviewed by Emmons and Sternberg, 1997). The SPV sensory neurons appear to inhibit transfer, so the signal presumably acts to inhibit the action of SPV. The SPD sensory neurons, on the other hand, play a positive role, perhaps to signal that spicule insertion has occurred (Liu and Sternberg, 1995). The male wiring project (www.wormatlas.org.) has recently shown that the SPCs, as well as the PCB and PCC sensory-motor neurons, innervate the gonad. It is interesting in this respect that all three of these cholinergic neurons also function in spicule insertion (Garcia et al., 2001), making them prime candidates for a role in the coordination of spicule insertion and ejaculation. The vas deferens of higher eukaryotes convey sperm from the testis to the exterior and are composed of smooth muscle. The smooth muscle of the rat vas deferens has a complex endomembrane system that contains high levels of IP₃Rs (Villa et al., 1993), which appear to function both in the modulation of Ca²⁺ sparks, and as a leak pathway to limit the Ca²⁺ content of stores (White and McGeown, 2003). ITR-1 may play similar functions in regulating the contraction of the vas deferens through the activation of EGL-8, but it is also possible that they play a role in the upstream neuronal signals that trigger contraction.

Two of the three N-terminal ITR-1 variants are expressed in the vas deferens, this is under the control of promoter pB and pC. Given that the spermatheca and the vas deferens are the only two tissues to express these same two N-terminal ITR-1 variants, it is interesting to note the similarity in structure between the two. Rhodamine-phalloidin staining of the spermatheca reveals circumferentially arranged actin microfilaments (McCarter et al., 1997) that undergo peristaltic constrictive and dilatory behavior like epithelial smooth muscle (McCarter et al., 1999). itr-1 functions downstream of let-23 (encoding an EGF receptor) in the regulation of spermathecal dilations (Clandinin et al., 1998). Given that two ITR-1 variants are coexpressed in the spermatheca it would seem likely that their combined effect is required during spermathecal dilation, and the same combination is presumably required for sperm transfer in the vas deferens. It will be very interesting to investigate whether itr-1 has parallel functions in the spermatheca and vas deferens. Until the structure of the vas deferens is further characterized, this question will be difficult to answer, although genetic analysis of males using genes known to function in the spermathecal pathway may provide some information.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
We are grateful to Andy Fire's laboratory for GFP reporter and RNAi vectors, to René García for helpful discussions and sharing unpublished data, and to Arundhati Sengupta-Ghosh and Sarah Farmer for contributions made during undergraduate projects in our laboratory. Some nematode strains used in this study were supplied by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health National Center for Research Resources. This work was supported by the Medical Research Council and the Biotechnology and Biological Sciences Research Council. H.A.B. is an MRC Senior Fellow.

	Footnotes

 
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E05–02–0096) on June 15, 2005.

Abbreviations used: IP₃, inositol 1,4,5-trisphosphate; IP₃R, inositol 1,4,5-trisphosphate receptor; PLC, phospholipase C; GFP, green fluorescent protein; ACh, acetylcholine; RNAi, RNA-mediated interference.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

These authors contributed equally to this work.

Address correspondence to: Howard A. Baylis (hab{at}mole.bio.cam.ac.uk).

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