Elsevier

Autonomic Neuroscience

Volume 84, Issue 3, 1 November 2000, Pages 147-161
Autonomic Neuroscience

P2X2 receptor expression by interstitial cells of Cajal in vas deferens implicated in semen emission

https://doi.org/10.1016/S1566-0702(00)00200-9Get rights and content

Abstract

Male reproduction is dependent upon seminal emission mediated by vas deferens contraction. This drives spermatic fluid to the prostatic urethra during ejaculation. We localize interstitial cells of Cajal (ICC), which express P2X2 receptor, subunits of ATP-gated ion channels, to rat, mouse and guinea-pig vas deferens submucosa. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of rat vas deferens resolved two functional splice variant transcripts of the P2X2 receptor subunit. The P2X2 receptor mRNA was localized principally within the lamina propria (submucosal) region of the rat vas deferens using in situ hybridization (ISH) and in situ RT-PCR-ISH. Immunohistochemistry using rat, mouse and guinea-pig vas deferens tissues confirmed expression of P2X2 receptor protein within the lamina propria, particularly within a dense column of small spindle-shaped cells adjacent to the columnar epithelial cells which line the lumen. This immunoreactivity was co-localized with neurone-specific enolase (NSE) and c-Kit protein expression, gene markers for ICC. Mucosal mast cells were distinguished from ICC by toluidine blue staining. Choline acetyltransferase immunoreactivity, a marker for post-ganglionic parasympathetic innervation, occurred on the lateral margin of the lamina propria and extended into the inner longitudinal muscle layer. P2X1 receptor immunolabelling was associated with sympathetic innervation of the smooth muscle in the outer longitudinal and circular muscle layers, but not the inner longitudinal layer. The physiological significance of the vas deferens ICC which express P2X2 receptors remains to be established. Possible roles include regulation of smooth muscle activity or mucosal secretion utilizing local ATP signaling, both of which would affect semen transport.

Introduction

Seminal release is divided into emission and ejaculation. Emission is the pulsatile transport of semen from storage in the epididymus through the lumen of the vas deferens to the prostatic urethra (Kolbeck and Steers, 1992). Neural stimulation of the vas deferens induces a biphasic contraction of smooth muscle leading to seminal emission (de Groat and Booth, 1980, Steers, 1994, Sneddon et al., 1996). Innervation of the vas deferens is derived from both the sympathetic and parasympathetic nervous systems. The majority of vas deferens neural input arises from sympathetic pre-ganglionic neurones in the lumbar spinal cord which synapse with post-ganglionic neurones in the pelvic accessory ganglion to innervate the vas deferens via the hypogastric nerve (Keast, 1992, Kolbeck and Steers, 1993, Kaleczyc et al., 1997). Parasympathetic pre-ganglionic cholinergic neurones originate from the lumbosacral spinal cord and traverse the major pelvic ganglion to innervate the vas deferens (Kolbeck and Steers, 1993, Kujat et al., 1993).

A small number of afferent fibres from the vas deferens travel through the hypogastric and pelvic nerves to the lumbosacral dorsal horn of the spinal cord (Kolbeck and Steers, 1992, Kolbeck and Steers, 1993). The role of afferents supplying the vas deferens is thought to relate to nociceptive or mechanoreceptive function (Floyd et al., 1976, Kolbeck and Steers, 1992).

The lumen of the vas deferens is surrounded by a columnar epithelial layer, a lamina propria, and three layers of smooth muscle fibres differentiated by orientation. This tissue is highly innervated, with muscle fibres adjacent to nerve varicosities (Burnstock, 1986). The majority of the post-ganglionic sympathetic fibres target the muscle layers, post-ganglionic parasympathetic (cholinergic) fibres target the lamina propria region and the inner longitudinal muscle layer. Peptidergic fibres target both regions (Keast, 1992). Sympathetic stimulation results in the co-release of noradrenaline and ATP (Vizi and Burnstock, 1988, Goncalves et al., 1995, Sneddon et al., 1996). Physiological experiments have shown that while noradrenaline mediates the secondary prolonged contraction phase, ATP is responsible for the initial rapid contraction of the vas deferens via P2X receptor activation (Friel, 1988, von Kügelgen and Starke, 1991, Burnstock, 1995, Sneddon et al., 1996, Nakanishi et al., 1997, Guitart et al., 1999, O’Connor et al., 1999).

P2X receptor subunits assemble to form homomeric or heteromeric ATP-gated ion channels (Brake et al., 1994, Valera et al., 1994, Lewis et al., 1995, Lê et al., 1998). The P2X1 receptor (P2X1R) was originally identified with the expression cloning of P2X1R cDNA from rat vas deferens (Valera et al., 1994). Northern blot analysis localized P2X1R mRNA expression within smooth muscle, neurones of the spinal cord and neonatal brain (Collo et al., 1996). Immunohistochemical analysis localized the presence of P2X1R expression to the outer longitudinal and circular muscle layers of vas deferens (Vulchanova et al., 1996, Worthington et al., 1999, Mulryan et al., 2000). Knock-out of the P2X1R subunit in mice significantly reduced sympathetic nerve-induced contraction of vas deferens, and induced male sterility by reducing the sperm count in the ejaculate, suggesting application in male contraception (Mulryan et al., 2000). Expression of the mRNA of P2X2R and P2X4R receptors subunits has also been detected by Northern blotting and reverse transcription polymerase chain reaction in vas deferens (Brake et al., 1994, Bo et al., 1995, Housley et al., 1995).

Here we report the localization of P2X2R mRNA and protein expression principally within the lamina propria region. We provide immunohistochemical evidence that the cells expressing the P2X2R subunit are interstitial cells of Cajal. The potential physiological significance of these findings is considered.

Section snippets

Materials and methods

Vas deferens tissue obtained from adult male Wistar strain rats, Swiss CD-1 strain mice and English short-hair guinea-pigs killed using pentobarbitone (Nembutal, 120 mg/kg, Virbac). Procedures followed guidelines approved by University of Auckland, Animal Ethics Committee.

Molecular characterization of P2X2 receptor mRNA isoforms within rat vas deferens

Two P2X2R subunit mRNA isoforms were isolated from rat vas deferens tissue. RT-PCR using mRNA from vas deferens and P2X2R specific primers yielded two distinct PCR products which migrated at approximately 700 and 480 bp with agarose gel electrophoresis (Fig. 1). Sequencing of the cloned PCR products indicated that these cDNAs were the P2X2–1R isoform described in PC12 cells (GenBank accession No. U14414) (Brake et al., 1994) and the P2X2–2R isoform which lacks 207 bp in the C-terminal coding

Discussion

Interstitial cells of Cajal (ICC) were resolved as a dense clustering of small cells with extensive reticular processes within the submuscosal (lamina propria) region of rat, mouse and guinea-pig vas deferens by neuron-specific enolase and c-Kit immunolabelling. This cell type expressed very high levels of the P2X2R subunit in all three species of animals examined. These P2XR subunits assemble in the cell membrane to form non-selective cation channels gated by extracellular ATP. The study

Acknowledgements

Supported by the Auckland Medical Research Foundation, the Marsden Fund and the New Zealand Lottery Grants Board.

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