Elsevier

Neuroscience

Volume 108, Issue 2, 10 December 2001, Pages 331-340
Neuroscience

Testosterone and nerve growth factor have distinct but interacting effects on structure and neurotransmitter expression of adult pelvic ganglion cells in vitro

https://doi.org/10.1016/S0306-4522(01)00420-1Get rights and content

Abstract

Circulating testosterone has potent effects on the structure and function of many pelvic ganglion cells in adult rats in vivo. However not all androgen-sensitive pelvic neurones possess androgen receptors and testosterone effects may therefore be indirect, by an action on the target organs. Here we have examined if testosterone influences neuronal structure in vitro in pelvic ganglion cells cultured from adult male rats. We have also used multiple label immunofluorescence to monitor the expression of transmitter-synthesising enzymes and peptides under various culture conditions. Testosterone was a more potent stimulant of noradrenergic soma growth in culture than nerve growth factor. Whereas nerve growth factor increased the number, branching and length of neurites, testosterone stimulated growth of a small number of very short processes, each of which bore numerous short protrusions. Testosterone also impeded the longer neurite growth induced by nerve growth factor. Many pelvic ganglion cells altered their expression of transmitters/neuropeptides under different culture conditions. In particular, under control conditions or during nerve growth factor treatment, vasoactive intestinal peptide was up-regulated in noradrenergic and cholinergic neurones; testosterone impeded this up-regulation in noradrenergic neurones. Choline acetyltransferase immunoreactivity could only be visualised when nerve growth factor was present in the cultures, and cholinergic neurones showed less neurite outgrowth than noradrenergic neurones under all culture conditions. Nerve growth factor did not stimulate levels of this enzyme as strongly if testosterone was present.

This study has shown that testosterone has potent effects on the structure of many pelvic ganglion cells in vitro. It is possible that these effects are mediated indirectly, e.g. by stimulating glial-derived substances, however our results suggest that the effects are not mediated by nerve growth factor. The results also show that testosterone influences some of the actions of nerve growth factor, suggesting that there may be complex interactions between steroid signalling and neurotrophic factors in maintaining neuronal structure and function in vivo.

Section snippets

Experimental procedures

Pelvic ganglia were removed from 41 male outbred Wistar rats (7 weeks of age) anaesthetised with sodium pentobarbitone (48 mg/kg) and animals were then killed. Our procedure did not cause animal suffering and was approved by the institutional animal ethics committee of the University of Queensland, complying with the Australian code of practice for the care and use of animals for scientific purposes. The number of animals used was the minimum required for reliable scientific data. Ganglion

General features of cultured cells

Many neurones started to form neurites after the first day in culture and growth continued in the second day. However in untreated cultures, some neurones did not form neurites even after 2 days such that the mean number of neurites was less than one (Fig. 1, Fig. 3). Under these conditions, most neurites were short (<10 μm) and unbranched. Each treatment had distinct effects on neurite outgrowth (see below), but did not appear to influence the number of neurones surviving on each coverslip.

Discussion

This study has shown for the first time that testosterone can induce significant structural and chemical changes in adult autonomic ganglion cells in vitro. The most profound effects occur in soma size and transmitter expression, and the former mimic those seen in vivo. The changes do not resemble those induced by NGF, indicating that effects of testosterone in vivo are unlikely to mediated by this neurotrophin. However, testosterone inhibits some of the NGF effects, suggesting a level of

Acknowledgements

This study was supported by the National Health and Medical Research Council (Australia), Grant numbers 34487 and 9935774.

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