Sexual differentiation of the spinal nucleus of the bulbocavernosus is not mediated solely by androgen receptors in muscle fibers

Abstract

The spinal nucleus of the bulbocavernosus (SNB) neuromuscular system is a highly conserved and well-studied model of sexual differentiation of the vertebrate nervous system. Sexual differentiation of the SNB is currently thought to be mediated by the direct action of perinatal testosterone on androgen receptors (ARs) in the bulbocavernosus/levator ani muscles, with concomitant motoneuron rescue. This model has been proposed based on surgical and pharmacological manipulations of developing rats as well as from evidence that male rats with the testicular feminization mutation (Tfm), which is a loss of function AR mutation, have a feminine SNB phenotype. We examined whether genetically replacing AR in muscle fibers is sufficient to rescue the SNB phenotype of Tfm rats. Transgenic rats in which wild-type (WT) human AR is driven by a human skeletal actin promoter (HSA-AR) were crossed with Tfm rats. Resulting male HSA-AR/Tfm rats express WT AR exclusively in muscle and nonfunctional Tfm AR in other tissues. We then examined motoneuron and muscle morphology of the SNB neuromuscular system of WT and Tfm rats with and without the HSA-AR transgene. We observed feminine levator ani muscle size and SNB motoneuron number and size in Tfm males with or without the HSA-AR transgene. These results indicate that AR expression in skeletal muscle fibers is not sufficient to rescue the male phenotype of the SNB neuromuscular system and further suggest that AR in other cell types plays a critical role in sexual differentiation of this system.

Figure 1

HSA-AR mRNA and protein expression in WT and Tg rats. A, RT-PCR products for HSA-AR mRNA expression in muscle visualized on an agarose gel. HSA-AR expression was detectable in Tg neonatal limb and BC/LA muscles and adult LA muscle but not WT muscles. B, Western immunoblot analysis shows AR overexpression for both the EDL and LA muscles (mean ± sem; n = 5 for Tg LA, WT EDL, and WT LA; n = 4 for Tg EDL). Graph represents mean ± sem. **, P < 0.01; ***, P < 0.001.

Figure 2

An increase in AR protein was found in limb muscle fibers for neonatal HSA-AR Tg rats. Photomicrographs show immunoreactivity for basal lamina only (red; A and D), AR only (green; B and E), and combined basal lamina and AR (C and F) for Tfm/WT (A, B, C) and Tfm/HSA-AR (D, E, and F) rats. Scale bar, 40 μm.

Figure 3

HSA-AR functionality. A, Increased myoglobin mRNA expression was found in adult HSA-AR Tg EDL muscle in comparison with WT muscle using quantitative PCR. B, Increased LBM (percent) was found for HSA-AR Tg rats on both WT and Tfm backgrounds. Graphs represent mean ± sem. *, P < 0.05; ***, P < 0.001.

Figure 4

Hematoxylin and eosin-stained sections through the perineum of PND 1 rats showing obvious sex differences in LA muscle size for a representative WT male (A), WT female (B), Tfm/WT male (C), and Tfm/HSA-AR male (D). Scale bar, 500 μm. R, Rectum.

Figure 5

Quantification of LA muscle volume in neonatal Tfm rats with and without the HSA-AR transgene. A, On PND 1, LA volume was larger in WT males than WT females and Tfm males with and without the transgene, and the transgene had no effect on LA size in Tfm males (n = 6). B, On PND 2.5, after perinatal testosterone propionate treatment, LA volumes were larger in WT males (n = 4) and females (n = 5) than Tfm males (n = 6), and there was no effect of the transgene on LA volume. Graphs represent mean ± sem. **, P < 0.01; ***, P < 0.001.

Figure 6

Quantification of motoneuron number and soma size for adult WT and Tfm male rats with and without the HSA-AR transgene. Motoneurons in the SNB were more numerous (A) and larger (B) in WT males than Tfm males, and there was no effect of the transgene. Motoneurons in the RDLN did not differ between groups for either number (C) or soma size (D). Graphs represent mean ± sem. ***, P < 0.001.