The proctodeum (the male rectum) includes a pair of prong-like sensory structures called the copulatory spicules (MaleSpicFIG 1; MaleSpicFIG 2; MaleSpicTABLE 1). The spicules are used by the male during mating to probe for the vulval opening and to lock onto the vulva and and hold it open during ejaculation to aid in sperm transfer (Liu and Sternberg, 1995; Garcia et al, 2001). Five muscles are attached to each of these spicules (2 retractor muscles hold spicules inisde male tail, two protractor muscles, each of which is innervated by an SPC motor neuron, force it out of the cloaca during mating. The anal depressor muscle reorganizes and forms an auxiliary extension to both the left and right protractor muscles during L4 (see below and Male Specific Muscles chapter).

Each of the left and right spicules consists of a sclerotized cuticle shell that encases the dendrites of 2 sensory neurons, SPDL/R and SPVL/R (MaleSpicFIG 3). The neuron processes are surrounded by spicule sheath cell (SPsh) syncytium which, in turn, is surrounded by spicule socket cell (SPso) syncytium (MaleSpicFIG 4, MaleSpicFIG 6). The sheath syncytium is a fusion of two cells (SPshD/VL or SPshD/VR), the socket syncytium a fusion of 4 cells (SPso1-4L or SPso1-4R) (MaleSpicFIG 5; MaleSpicTABLE 1). The socket cells secrete a cuticle that covers the outside of the spicule (Sulston et al., 1980; Jiang and Sternberg, 1999). The sensory endings of the neurons are exposed at the spicule tip and so possibly sense chemosensory cues on the hermaphrodite surface during spicule prodding or in the hermaphrodite uterine environment during spicule insertion. The cell bodies of spicule neurons are located in the male-specific cloacal ganglia (L/R), which flank the proctodeum. SPsh and SPso cell bodies lie more anteriorly (MaleSpicFIG 3; MaleSpicFIG 6; Sulston et al., 1980).

Spicules are also associated with a third pair of neurons, the sensory-motor neurons SPCL/R (MaleSpicFIG 3). In contrast to SPD and SPV neurons, SPCL/R do not enter the spicules. They are attached to and innervate the protractor muscles that surround the spicules and control spicule movement (MaleSpicFIG 6B, MaleSpicFIG 7A). The sensory endings of SPCL/R are attached directly to the dorsal proctractor muscle via hemidesmosomes (Hd) (MaleSpicFIG 6B). Their commissures form NMJs with dorsal and ventral protractors as they pass between them (MaleSpicFIG 7B; Sulston et al., 1980; The Male Wiring Project).

2 Spicule Neuron Connectivity

Major synaptic targets of the spicule neurons include each other, hook and PCS neurons, the protractor muscles and the gonad (The Male Wiring Project). These connections emphasize that spicule behavior must be coordinated with the preceding step of vulval location and the subsequent step of sperm transfer (reviewed in WormBook: Male Mating Behavior - Barr and Garcia, 2006). During mating the hook and PCS detect the general location of the vulva and trigger spicule prodding behavior by inducing periodic contraction of the spicule protractor muscles (MaleSpicMOVIE 1). This stimulation appears to be indirect as neither hook nor PCS neurons synapse onto the muscles. SPCL/R, having both proprioceptive and motor connections with the protractor muscles, register when the vulval slit is breached and induce tonic muscle contraction and full spicule insertion (MaleSpicFIG 6B; MaleSpicFIG 7B; Garcia et al, 2001). Sensory neurons SPVL/R are required to inhibit premature ejaculation (Liu and Sternberg, 1995) but surprisingly do not have direct connections to the gonad. These neurons may therefore exert their effect through their connections with SPCL/R and the PCS neurons which innervate vas deferens and cloacal cells in the gonad-cloacal junctional region (MaleSpicFIG 7C). SPDL/R connectivity are not known however they are the major targets of SPVL/R.

All spicule cells, including spicule neurons, are derived from the rectal cell B which is present in the L1 of both sexes but is a blast cell only in the male (MaleSpicFIG 8; Sulston and Horvitz, 1977; Sulston et al., 1980). In males, B generates 47 progeny which form the cloaca and spicule channels that house the spicules, as well as the spicules themselves. Spicule development can be divided into 2 phases (1) spicule cell generation and (2) spicule morphogenesis.

The B cell lineage is defined by three distinct phases: early division, short range migrations and late divisions (MaleSpicFIG 8). The early divisions begin in late L1 where the B cell divides asymmetrically to produce a large anterior daughter (B.a) and a small posterior daughter (B.p). This asymmetry is regulated by the Wnt pathway (a LIN-44 /LIN-17 pathway; Sternberg and Horvitz, 1988; Herman and Horvitz, 1994). By mid-L2 B.a and B.p produce a total of 10 cells (MaleSpicFIG 8, MaleSpicFIG 9): B.a generates 4 cell pairs and B.p 1 pair. The 4 B.a-derived pairs migrate a short distance, arranging themselves in 2 rings of 4 cells around the developing cloaca. Among the aa and pp cells migration of the left and right cell is variable; either the left or right member can take the anterior position near the cloaca. Signals from neighboring cells of the F, U (EGF LIN-3/ LET-23 signaling) and Y lineages (LIN-15) promote differences between these otherwise equivalent cells causing them adopt an anterior fate (for aa's = α, for pp's=γ) or a posterior fate (for aa's = β; for pp's= δ) (distinguished by the lineage produced in later cell divisions; Chamberlin and Sternberg, 1993; 1994). Thus, the B lineage represents one of the few cases in C. elegans where cell fate is not fixed by lineal origin and is determined by environmental cues. Cells of the B.p lineage are patterned by Notch signaling (LIN-12) (MaleSpicFIG 8; Chamberlin and Sternberg, 1994). During L3, precursors α, β, ζ and γ generate the cells of the spicules.

The B cell lineage is complete by the L3/L4 molt. Spicule morphogenesis occurs during L4 and is part of a larger process of tail remodeling that begins at mid-L4 (38-40hrs, 20°C) and ends by the final molt (45hrs) (MaleSpicFIG 9; MaleSpicFIG 10; MaleSpicFIG 11; MaleSpicFIG 12); for tail remodeling see Epithelial System of the Male - Hypodermis). In the adult, 2 pairs of proctodeal cells, Bε(l/r).pv (aka B.a(l/r)appv) and Bε(l/r).apa (B.a(l/r)apapa), lie immediately dorsal and ventral of the spicules and line the spicule channels (MaleSpicFIG 10). These cells play a major role in establishing the characteristic elongated morphology of the spicules and the channel. During morphogenesis Bε(l/r).pv and Bε(l/r).apa migrate anteriorly, establishing cellular molds (spicule traces) into which future spicule cuticle is secreted (MaleSpicFIG 10). Their migration is dependent on a TGF-β (DBL-1) signal, possibly coming from dorsal sex muscles (Baird and Ellazar, 1999).

Bε(l/r).pv insert "fingers" into the dorsal spicule retractor (dsr) muscles (MaleSpicFIG 10; see also MaleProcFIG 7 in Epithelial System of the male - Proctodeum). The forward movement of retractors during tail morphogenesis likely facilitates spicule trace elongation. An FGF signal (EGL-17), expressed in the SPso cells (MaleSpicFIG 11), has also been implicated in the process (Jiang and Sternberg, 1999; reviewed in WormBook: Male Development - Emmons). The SPso cells are essential for spicule elongation and also for secretion of the spicule cuticle, which begins at ca. 42 hrs (Jiang and Sternberg, 1999). Both spicule elongation and cuticle formation depend on the activity of heterochronic transcription factor LIN-29 which is expressed in B lineage cells (Euling et al., 1999).

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