Effects of Bax Gene Deletion on Muscle and Motoneuron Degeneration in a Sexually Dimorphic Neuromuscular System

Abstract

Motoneurons in the spinal nucleus of the bulbocavernosus (SNB) and their target muscles in the perineum, bulbocavernosus (BC), and levator ani (LA) normally degenerate in female rodents. Death of the motoneurons and muscles can be prevented by androgen treatments around the time of birth. To identify the intracellular mechanisms underlying hormone-dependent survival of this neuromuscular system, we examined mice with a targeted disruption of the pro-death gene Bax. SNB motoneuron number was increased in female Bax؊/؊ mice, whether measured using immunolabeling for a motoneuron-specific marker or retrograde labeling with the fluorescent tracer Fluoro-Gold. Based on retrograde tracing, the sex difference in SNB cell number is eliminated in Bax؊/؊ mice. Thus, Bax is required for sexually dimorphic motoneuron death in the SNB, and motoneurons rescued by Bax deletion project their axons to the periphery. Mean soma size in the SNB of Bax؊/؊ females is reduced, however, and there is a subpopulation of very small cells in the SNB of female knock-outs. In addition, the BC muscle was not identified in any female, regardless of Bax gene status. All females possessed a small LA muscle, and Bax deletion resulted in a tripling of LA fiber number in females. This increase was small, however, relative to the Ͼ50-fold sex difference in LA muscle fiber number. Thus, the sex difference in the perineal muscles is mostly unaffected by the absence of Bax protein, and SNB motoneuron number is dissociated from target muscle size in Bax؊/؊ animals.

Neuromuscular Development in the Absence of Programmed Cell Death: Phenotypic Alteration of Motoneurons and Muscle

“…Fluorogold injections into the whisker pads labeled significantly more facial MNs in the Bax KO (30%; p Ͻ 0.01) (data not shown), suggesting that many of the excess facial MNs innervated their targets (see also Jacob et al, 2005). Although the significant increase in retrogradely labeled facial MNs in the Bax KO suggests that many rescued cells may have formed synaptic contacts, because we did not attempt to directly quantify neuromuscular (2) n.d.…”

“…Previous studies of motor systems developing with reduced PCD have reported more cell bodies in central motor nuclei or more axons in cranial or spinal roots , but with the exception of paralytic chicks and mice (Ding et al, 1983;Oppenheim and Chu-Wang, 1983;Landmesser, 1992;Misgeld et al, 2002), whether rescued MNs project to their muscle targets has not been systematically examined Jacob et al, 2005). In cross sections of adult peripheral hindlimb and cranial nerves (mixed motor and sensory) of Bax KO and MyoGDNF mice (and in the paralyzed embryonic chick), we observed more (ϳ 40 -50%) myelinated axons in close proximity to their muscles compared with WT mice or vehicle-treated chick embryos (Table 1, Fig.…”

“…Since the initial realization that PCD is a natural developmental process, there has been an increasing interest in elucidating its molecular regulation, but less interest in its biological utility. An excess of trophic molecules (Oppenheim, 1996;Oppenheim et al, 2000), the arrest of embryonic movements by neuromuscular activity blockade (Houenou et al, 1990;Oppenheim, 1991;Oppenheim et al 2000;Misgeld et al 2002;Terrado et al, 2001;Brandon et al, 2003), and genetic perturbation of apoptotic biochemical pathways (Knudson et al, 1995;White et al, 1998;Jacob et al, 2005) have all been shown to significantly reduce or completely prevent developmental PCD of select neuronal populations, including motoneurons (MNs).…”

The spinal nucleus of the bulbocavernosus: Firsts in androgen-dependent neural sex differences

“…Although an LA muscle was present in every adult female Great Wistar rat examined (Tobin and Joubert, 1988), no LA fibers were found in the majority of Sprague Dawley rats examined, based on microscopic analysis of serial sections through the perineum (Bengston et al, 1996). In mice, the LA of adult females is miniscule, averaging only about 50 fibers (Jacob et al, 2004). Moreover, although cell addition and growth undoubtedly contribute to the sex difference in LA size, several observations suggest that cell death also plays an important role in sexual differentiation of both the LA and BC muscles.…”

“…We find TUNEL-positive cells in the BC and LA muscles of both sexes perinatally, but the density of apoptotic cells is significantly greater in females (Figure 4; Jacob and Forger, unpublished). Moreover, LA muscle fiber number is increased in female mice lacking the pro-death gene, Bax (Jacob et al, 2004), and is massively increased in double knockout mice lacking Bax and Bak, two genes critical for cell death in many tissues (Jacob, Ray, Lindsten and Forger, unpublished). Thus, cell death contributes to sexual differentiation of the LA, at least in mice.…”