Testosterone metabolites differentially maintain adult morphology in a sexually dimorphic neuromuscular system

The lumbar spinal cord of rats contains the sexually dimorphic, steroid‐sensitive spinal nucleus of the bulbocavernosus (SNB). Androgens are necessary for the development of the SNB neuromuscular system, and in adulthood, continue to influence the morphology and function of the motoneurons and their target musculature. However, estrogens are also involved in the development of the SNB system, and are capable of maintaining function in adulthood. In this experiment, we assessed the ability of testosterone metabolites, estrogens and nonaromatizable androgens, to maintain neuromuscular morphology in adulthood. Motoneuron and muscle morphology was assessed in adult normal males, sham‐castrated males, castrated males treated with testosterone, dihydrotestosterone, estradiol, or left untreated, and gonadally intact males treated with the 5α‐reductase inhibitor finasteride or the aromatase inhibitor fadrozole. After 6 weeks of treatment, SNB motoneurons were retrogradely labeled with cholera toxin‐HRP and reconstructed in three dimensions. Castration resulted in reductions in SNB target muscle size, soma size, and dendritic morphology. Testosterone treatment after castration maintained SNB soma size, dendritic morphology, and elevated target muscle size; dihydrotestosterone treatment also maintained SNB dendritic length, but was less effective than testosterone in maintaining both SNB soma size and target muscle weight. Treatment of intact males with finasteride or fadrozole did not alter the morphology of SNB motoneurons or their target muscles. In contrast, estradiol treatment was completely ineffective in preventing castration‐induced atrophy of the SNB neuromuscular system. Together, these results suggest that the maintenance of adult motoneuron or muscle morphology is strictly mediated by androgens. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 70: 206–221, 2010.     

Castration‐induced upregulation of muscle ERα supports estrogen sensitivity of motoneuron dendrites in a sexually dimorphic neuromuscular system

The spinal cord of rats contains the sexually dimorphic motoneurons of the spinal nucleus of the bulbocavernosus (SNB). In males, SNB dendrites fail to grow after castration, but androgen or estrogen treatment supports dendritic growth in castrated males. Estrogenic support of SNB dendrite growth is mediated by estrogen receptors (ER) in the target muscle. ERα expression in cells lacking a basal lamina (referred to as “extra‐muscle fiber cells”) of the SNB target musculature coincides with the period of estrogen‐dependent SNB dendrite growth. In the SNB target muscle, extra‐muscle fiber ERα expression declines with age and is typically absent after postnatal (P) day 21 (P21). Given that estradiol downregulates ERα in skeletal muscle, we tested the hypothesis that depleting gonadal hormones would prevent the postnatal decline in ERα expression in the SNB target musculature. We castrated male rats at P7 and assessed ERα immunolabeling at P21; ERα expression was significantly greater in castrated males compared with normal animals. Because ERα expression in SNB target muscles mediates estrogen‐dependent SNB dendrogenesis, we further hypothesized that the castration‐induced increase in muscle ERα would heighten the estrogen sensitivity of SNB dendrites. Male rats were castrated at P7 and treated with estradiol from P21 to P28; estradiol treatment in castrates resulted in dendritic hypertrophy in SNB motoneurons compared with normal males. We conclude that early castration results in an increase in ERα expression in the SNB target muscle, and this upregulation of ERα supports estrogen sensitivity of SNB dendrites, allowing for hypermasculinization of SNB dendritic arbors. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 921–935, 2013     

Critical period for estrogen‐dependent motoneuron dendrite growth is coincident with ERα expression in target musculature

The spinal cord of rats contains the sexually dimorphic, steroid‐sensitive motoneurons of the spinal nucleus of the bulbocavernosus (SNB). In males, SNB dendrite growth is dependent on gonadal steroids: dendrite growth is inhibited after castration, but supported in androgen‐ or estrogen‐treated castrated males. Furthermore, estrogenic support of SNB dendrite growth is mediated by estrogen action at the target musculature, inhibited by estrogen receptor (ER) blockade at the muscle and supported by local estradiol treatment. However, this estrogenic support is restricted to the early postnatal period, after which the morphology of SNB dendrites is insensitive to estrogens. To test if the developmentally restricted effects of estrogens on SNB dendrite growth coincide with the transient expression of ER in the target musculature, ERα expression was assessed during development and in adulthood. ERα expression in extra‐Muscle fiber cells was greatest from postnatal day 7 (P7) to P14 and declined after P21. Because this pattern of ERα expression coincided with the period of estrogen‐dependent dendrite growth, we tested if limiting hormone exposure to the period of maximal ERα expression in extra‐muscle fiber cells could fully support estrogen‐dependent SNB dendrite growth. We restricted estradiol treatment in castrated males from P7 to P21 and assessed SNB dendritic morphology at P28. Treating castrates with estradiol implants at the muscle from P7 to P21 supported dendrite growth to normal levels through P28. These data suggest that the transient ERα expression in target muscle could potentially define the critical period for estrogen‐dependent dendrite growth in SNB motoneurons. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Androgen action at the target musculature regulates brain‐derived neurotrophic factor protein in the spinal nucleus of the bulbocavernosus

We have previously demonstrated that brain‐derived neurotrophic factor (BDNF) interacts with testosterone to regulate dendritic morphology of motoneurons in the highly androgen‐sensitive spinal nucleus of the bulbocavernosus (SNB). Additionally, in adult male rats testosterone regulates BDNF in SNB motoneurons and its target muscle, the bulbocavernosus (BC). Because BDNF is retrogradely transported from skeletal muscles to spinal motoneurons, we hypothesized that testosterone could regulate BDNF in SNB motoneurons by acting locally at the BC muscle. To test this hypothesis, we restricted androgen manipulation to the SNB target musculature. After castration, BDNF immunolabeling in SNB motoneurons was maintained at levels similar to those of gonadally intact males by delivering testosterone treatment directly to the BC muscle. When the same implant was placed interscapularly in castrated males it was ineffective in supporting BDNF immunolabeling in SNB motoneurons. Furthermore, BDNF immunolabeling in gonadally intact adult males given the androgen receptor blocker hydroxyflutamide delivered directly to the BC muscle was decreased compared with that of gonadally intact animals that had the same hydroxyflutamide implant placed interscapularly, or when compared with castrated animals that had testosterone implants at the muscle. These results demonstrate that the BC musculature is a critical site of action for the androgenic regulation of BDNF in SNB motoneurons and that it is both necessary and sufficient for this action. Furthermore, the local action of androgens at the BC muscle in regulating BDNF provides a possible mechanism underlying the interactive effects of testosterone and BDNF on motoneuron morphology. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 587–598, 2013     

Aromatase inhibition reduces dendritic growth in a sexually dimorphic rat spinal nucleus

The rat lumbar spinal cord contains the steroid-sensitive spinal nucleus of the bulbocavernosus (SNB), whose motoneurons innervate perineal muscles involved in copulatory reflexes. In normal males, SNB motoneuron dendrites grow exuberantly through postnatal (P) day 28. This growth is steroid dependent: Dendrites fail to grow in males castrated at P7, but grow normally in castrates treated with testosterone or its metabolites, dihydrotestosterone combined with estrogen. Treatment with either metabolite alone supports dendritic growth, but not to the level of testosterone-treated or intact males. In this study, we tested the hypothesis that aromatization of androgens to estrogens was involved in the masculine development of SNB dendrites. Motoneuron morphology was assessed in normal males and males treated daily (P7-28) with fadrozole, a potent aromatase inhibitor (0.25 mg/kg, subcutaneously) or saline vehicle (n = 4-6/group). SNB motoneurons were retrogradely labeled with cholera toxin-horseradish peroxidase at P28 (when dendritic length is normally maximal) and reconstructed in three dimensions. Comparable labeling was seen across groups; it was equivalent in both the rostrocaudal and radial extents. However, dendritic lengths in fadrozole-treated males were significantly below those of intact or saline-treated males. Neither SNB somata size nor target muscle weight differed across groups. These results suggest that aromatization of androgens to estrogens is necessary for development of masculine SNB dendritic morphology.     

Differential effects of dihydrotestosterone and estrogen on the development of motoneuron morphology in a sexually dimorphic rat spinal nucleus

The rat lumbar spinal cord contains a sexually dimorphic motor nucleus, the spinal nucleus of the bulbocavernosus (SNB), whose motoneurons innnervate perineal muscles involved in copulatory reflexes. Dendritic development of SNB motoneurons is biphasic and androgen dependent. During the first 4 postnatal weeks, SNB dendrites grow exuberantly, and subsequently retract to mature lengths by 7 weeks of age. After early postnatal castration, SNB dendrites fail to grow, and testosterone replacement restores this growth. In other systems, testosterone and its metabolites, dihydrotestosterone and estrogen, are important for somatic and neural sexual differentiation. The purpose of the present study was to examine the effects of castration and dihydrotestosterone or estrogen replacement on the growth of SNB motoneuron somata and dendritic arbors. Male rat pups were castrated on postnatal (P) day 7 and treated daily with either dihydrotestosterone propionate (DHTP; 2 mg) or estradiol benzoate (EB; 100 μg) until P28 or P49. By using cholera toxin horseradish peroxidase (BHRP) histochemistry, the soma size, dendritic length, dendritic extent, and arbor area of BHRP-labeled SNB motoneurons were measured and analyzed. Both DHTP and EB treatment supported the initial exuberant growth of SNB dendrites through P28, but EB treatment was ineffective in maintaining mature, adult lengths at P49. The possible sites of hormone action and functional implications of these hormonal treatments are discussed. 1994 John Wiley & Sons, Inc.     

Ovariectomy attenuates dendritic growth in hormone‐sensitive spinal motoneurons

The lumbar spinal cord of rats contains the sexually dimorphic, steroid‐sensitive spinal nucleus of the bulbocavernosus (SNB). Dendritic development of SNB motoneurons in male rats is biphasic, initially showing exuberant growth through 4 weeks of age followed by a retraction to mature lengths by 7 weeks of age. The initial growth is steroid dependent, attenuated by castration or aromatase inhibition, and supported by hormone replacement. Dendritic retraction is also steroid sensitive and can be prevented by testosterone treatment, but is unaffected by aromatase inhibition. Together, these results suggest a role for estrogens during the initial growth phase of SNB development. In this study, we tested whether ovarian hormones could support SNB somal and dendritic development. Motoneuron morphology was assessed in normal males and in females perinatally masculinized with dihydrotestosterone and then either ovariectomized or left intact. SNB motoneurons were retrogradely labeled with cholera toxin‐HRP at 4 or 7 weeks of age and reconstructed in three dimensions. Initial growth of SNB dendrites was reduced after ovariectomy in masculinized females. However, no differences in dendritic length were seen at 7 weeks of age between intact and ovariectomized masculinized females, and lengths in both groups were significantly lower than those of normal males. Together with previous findings, these results suggest that estrogens are involved in the early growth of SNB dendrites, but not in their subsequent retraction. © 2001 John Wiley & Sons, Inc. J Neurobiol 48: 301–314, 2001     

Ovariectomy attenuates dendritic growth in hormone-sensitive spinal motoneurons

The lumbar spinal cord of rats contains the sexually dimorphic, steroid-sensitive spinal nucleus of the bulbocavernosus (SNB). Dendritic development of SNB motoneurons in male rats is biphasic, initially showing exuberant growth through 4 weeks of age followed by a retraction to mature lengths by 7 weeks of age. The initial growth is steroid dependent, attenuated by castration or aromatase inhibition, and supported by hormone replacement. Dendritic retraction is also steroid sensitive and can be prevented by testosterone treatment, but is unaffected by aromatase inhibition. Together, these results suggest a role for estrogens during the initial growth phase of SNB development. In this study, we tested whether ovarian hormones could support SNB somal and dendritic development. Motoneuron morphology was assessed in normal males and in females perinatally masculinized with dihydrotestosterone and then either ovariectomized or left intact. SNB motoneurons were retrogradely labeled with cholera toxin-HRP at 4 or 7 weeks of age and reconstructed in three dimensions. Initial growth of SNB dendrites was reduced after ovariectomy in masculinized females. However, no differences in dendritic length were seen at 7 weeks of age between intact and ovariectomized masculinized females, and lengths in both groups were significantly lower than those of normal males. Together with previous findings, these results suggest that estrogens are involved in the early growth of SNB dendrites, but not in their subsequent retraction.     

Castration reduces motoneuron soma size but not dendritic length in the spinal nucleus of the bulbocavernosus of wild-type and BCL-2 overexpressing mice

Motoneurons in the spinal nucleus of the bulbocavernosus (SNB) and their target muscles, bulbocavernosus and levator ani (BC/LA), constitute an androgen-sensitive neuromuscular system. Testosterone regulates SNB soma size, SNB dendritic length, and BC/LA muscle mass in adult male rats. Recent evidence indicates that the cell death-regulatory protein, Bcl-2, may also play a role in adult neural plasticity. The present study examined whether gonadal hormones and/or the Bcl-2 protein influence the morphology of the SNB neuromuscular system in adult B6D2F1 mice. In Experiment 1, adult wild-type and Bcl-2 overexpressing males were castrated and implanted with silastic capsules containing testosterone or left blank. Six weeks after castration, cholera toxin-horseradish peroxidase was injected into the BC muscle to label SNB dendrites. Animals were killed 48 h later, and BC/LA muscle mass, SNB soma size, and SNB dendritic arbors were examined. In Experiment 2, wild-type and Bcl-2 overexpressing males were castrated or sham castrated, implanted with testosterone-filled or blank capsules, and examined 12 weeks later. In both experiments, BC/LA muscle mass and SNB soma size were significantly reduced in castrates receiving blank capsules. Surprisingly, however, there was no effect of hormone manipulation on any of several measures of dendritic length. Thus, the dendritic morphology of SNB motoneurons appears to be relatively insensitive to circulating androgen levels in B6D2F1 mice. Bcl-2 overexpression did not influence BC/LA muscle mass, SNB soma size, or SNB dendritic length, indicating that the morphology of this neuromuscular system and the response to castration are not altered by forced expression of the Bcl-2 protein.     

Castration reduces motoneuron soma size but not dendritic length in the spinal nucleus of the bulbocavernosus of wild‐type and BCL‐2 overexpressing mice

Motoneurons in the spinal nucleus of the bulbocavernosus (SNB) and their target muscles, bulbocavernosus and levator ani (BC/LA), constitute an androgen‐sensitive neuromuscular system. Testosterone regulates SNB soma size, SNB dendritic length, and BC/LA muscle mass in adult male rats. Recent evidence indicates that the cell death‐regulatory protein, Bcl‐2, may also play a role in adult neural plasticity. The present study examined whether gonadal hormones and/or the Bcl‐2 protein influence the morphology of the SNB neuromuscular system in adult B6D2F1 mice. In Experiment 1, adult wild‐type and Bcl‐2 overexpressing males were castrated and implanted with silastic capsules containing testosterone or left blank. Six weeks after castration, cholera toxin‐horseradish peroxidase was injected into the BC muscle to label SNB dendrites. Animals were killed 48 h later, and BC/LA muscle mass, SNB soma size, and SNB dendritic arbors were examined. In Experiment 2, wild‐type and Bcl‐2 overexpressing males were castrated or sham castrated, implanted with testosterone‐filled or blank capsules, and examined 12 weeks later. In both experiments, BC/LA muscle mass and SNB soma size were significantly reduced in castrates receiving blank capsules. Surprisingly, however, there was no effect of hormone manipulation on any of several measures of dendritic length. Thus, the dendritic morphology of SNB motoneurons appears to be relatively insensitive to circulating androgen levels in B6D2F1 mice. Bcl‐2 overexpression did not influence BC/LA muscle mass, SNB soma size, or SNB dendritic length, indicating that the morphology of this neuromuscular system and the response to castration are not altered by forced expression of the Bcl‐2 protein. © 2002 Wiley Periodicals, Inc. J Neurobiol 53: 403–412, 2002     

Effects of testosterone on the development of neuromuscular systems and their target tissues involved in courtship and copulation in green anoles (Anolis carolinensis)

Male green anole lizards court females using a red throat fan (dewlap) and copulate by intromitting one of two penises (hemipenes). These structures begin sexually monomorphic, but by adulthood males have larger dewlaps, only males have hemipenes, and many of the neuromuscular components of both systems show male-biased dimorphisms. We hypothesized that testosterone (T), which increases in juvenile males but not females about a month after hatching, facilitates masculinization. To test this idea, on post-hatching day 30, gonadally intact females received either a blank or T implant, and males were either castrated or sham-castrated. At day 90, juveniles were euthanized and the length of the cartilage and cross-sectional areas of the muscle fibers and motoneurons required for dewlap extension were examined. We also measured the cross-sectional areas of the hemipenes and associated muscle fibers and motoneurons, and counted the motoneurons. T-treated females had longer cartilages and larger dewlap muscle fibers compared to those with blank implants. No effects on motoneurons were detected, and no females possessed hemipenes or associated musculature. In males, castration produced shorter dewlap cartilages and smaller hemipenes; other measures were not affected by treatment. These data indicate that components of the dewlap system differentiate relatively late in development, that T likely mediates the process, and that although components of the copulatory system are plastic in juvenile males, sexual differentiation of peripheral features is complete before day 30. The data also suggest that target structures (dewlap cartilage and hemipenes), compared to their neuromuscular effectors, are particularly sensitive to developmental T exposure.     

Restoration of male sexual behavior by adult exogenous estrogens in male aromatase knockout mice

We previously found that male aromatase knockout (ArKO) mice that carry a targeted mutation in exons 1 and 2 of the CYP19 gene and as a result cannot aromatize androgen to estrogen show impaired sexual behavior in adulthood. To determine whether this impairment was due to a lack of activation of sexual behavior by estradiol, we studied here male coital behavior as well as olfactory investigation of sexually relevant odors in male ArKO mice following adult treatment with estradiol benzoate (EB) or dihydrotestosterone propionate (DHTP). Again, we found that gonadally intact ArKO males show pronounced behavioral deficits affecting their male coital behavior as well as their olfactory investigation of volatile body odors but not that of soiled bedding. Deficits in male coital behavior were largely corrected following adult treatment with EB and the androgen DHTP, suggesting that estradiol has prominent activational effects on this behavior. By contrast, adult treatment with EB to either castrated or gonadally intact ArKO males did not stimulate olfactory investigation of volatile body odors, suggesting that this impairment may result from a lack of proper organization of this behavior during ontogeny due to the chronic lack of estrogens. In conclusion, the present studies suggest that the behavioral deficits in sexual behavior in male ArKO mice result predominantly from a lack of activation of the behavior by estrogens. This is in contrast with earlier pharmacological studies performed on rats and ferrets that have suggested strong organizational effects of estradiol on male sexual behavior.     

Estrogen alters excitability but not morphology of a sexually dimorphic neuromuscular system in adult rats

In rats, motoneurons of the spinal nucleus of the bulbocavernosus (SNB) innervate the bulbocavernosus (BC) muscle, which surrounds the base of the penis. The SNB/BC is a sexually dimorphic, steroid-sensitive neuromuscular system, which is critically important in male reproductive behavior. Androgens are necessary for the development, morphology, and function of the SNB/BC system. However, estradiol (E) is also necessary for the development of the SNB/BC system, and E is capable of maintaining BC EMG activity in adulthood. In this study, we used electrophysiological and anatomical methods to examine estrogenic effects on BC EMG activity. We used a modified H-reflex testing method to investigate polysynaptic reflex characteristics in intact males, castrates, and castrates treated short term with estradiol benzoate (EB). Measures of EMG activity, response latency, and spike count were altered in castrates, but maintained in EB-treated castrates to the levels of intact males. Furthermore, estrogenic effects were found in EMG activity that could be isolated to the periphery of the SNB/BC system. BC NMJ size and muscle fiber area have been demonstrated to be hormone sensitive, and we examined these for possible correlates of E's effects on BC EMG activity. BC muscles of intact males, castrates, and short-term EB-treated castrates were fixed and stained with zinc iodide and osmium tetroxide. NMJ size and muscle fiber area did not differ between groups. Together, these data suggest that E treatment results in changes in the neuromuscular periphery that maintain BC EMG activity, but this effect cannot be accounted for by changes in NMJ size or muscle fiber area.     

Adult plasticity in hormone-sensitive motoneuron morphology: methodological/behavioral confounds

Changes in androgen levels can alter the structure of motoneurons in the spinal nucleus of the bulbocavernosus (SNB), a motor nucleus that innervates perineal muscles involved in copulatory behavior. While sexual activity can alter androgen levels in normal males, it has no effect on SNB motoneuron soma size or dendritic morphology (Beversdorf, Kurz, and Sengelaub, 1990). However, Breedlove (1997) reported reductions in the size of SNB somata, nuclei, and target muscles of copulating versus noncopulating castrated rats maintained on subphysiological testosterone. To reconcile the results obtained using intact versus implant paradigms, we tested the hypothesis that the implant/behavior paradigm could produce differences in hormone levels, potentially confounding sexual behavior effects on the morphology of this androgen-sensitive neuromuscular system. Young adult male rats were castrated and immediately given 5-mm Silastic implants containing crystalline testosterone. One week later, blood samples were drawn and the males were housed with receptive females (copulators) or nonreceptive females (noncopulators) or housed alone (singles). After 27 days, blood samples were drawn again, and SNB target muscles and spinal cords removed. No differences in target muscle weight or SNB somata and nuclei size were observed between copulators, noncopulators, or singles; as expected, all measures were significantly reduced relative to intact males. Radioimmunoassay showed that testosterone declined differentially over the course of the behavioral manipulation across groups, being greatest in copulators and least pronounced in single males. These data indicate that differences in sexual or housing experience can alter testosterone titers under these implant conditions, potentially confounding hormone-sensitive measures of morphology.     

Androgenic, but not estrogenic, protection of motoneurons from somal and dendritic atrophy induced by the death of neighboring motoneurons

Motoneuron loss is a significant medical problem, capable of causing severe movement disorders or even death. We have been investigating the effects of motoneuron loss on surviving motoneurons in a lumbar motor nucleus, the spinal nucleus of the bulbocavernosus (SNB). SNB motoneurons undergo marked dendritic and somal atrophy following the experimentally induced death of other nearby SNB motoneurons. However, treatment with testosterone at the time of lesioning attenuates this atrophy. Because testosterone can be metabolized into the estrogen estradiol (as well as other physiologically active steroid hormones), it was unknown whether the protective effect of testosterone was an androgen effect, an estrogen effect, or both. In the present experiment, we used a retrogradely transported neurotoxin to kill the majority of SNB motoneurons on one side of the spinal cord only in adult male rats. Some animals were also treated with either testosterone, the androgen dihydrotestosterone (which cannot be converted into estradiol), or the estrogen estradiol. As seen previously, partial motoneuron loss led to reductions in soma area and in dendritic length and extent in surviving motoneurons. Testosterone and dihydrotestosterone attenuated these reductions, but estradiol had no protective effect. These results indicate that the neuroprotective effect of testosterone on the morphology of SNB motoneurons following partial motoneuron depletion is an androgen effect rather than an estrogen effect.     

Distribution of androgen receptor immunoreactivity in the spinal cord of wild-type,androgen-insensitive and gonadectomized male rats

The polyclonal antiserum PG21 was used to detect androgen receptor (AR) protein in three motoneuronal pools of the male rat lumbar spinal cord. In gonadally intact, wild-type males, the spinal nucleus of the bulbocavernosus (SNB), dorsolateral nucleus (DLN), and retrodorsolateral nucleus (RDLN) all displayed immunolabeling of cell nuclei. The percentage of motoneurons displaying such labeling was highest in the SNB and lowest in the RDLN. This pattern of AR immunocytochemical labeling agrees well with previous steroid autoradiographic studies of androgen accumulation in the rat spinal cord. In contrast, virtually no motoneurons in any of the three pools displayed nuclear AR immunostaining in long-term gonadectomized males or in gonadally intact males carrying the Tfm mutation, which renders the AR incompetent. In gonadectomized males, labeling was restored in the SNB and DLN, but not the RDLN, 30 min after an injection of replacement testosterone. Eight hours of testosterone exposure restored immunolabeling in all three motor nuclei. Apparent cytoplasmic staining was seen in SNB motoneurons of untreated castrates and Tfm rats, but not intact rats, suggesting that AR residing in the cytoplasm translocates to the nucleus on binding to androgen in these motoneurons. © 1995 John Wiley & Sons, Inc.     

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

Cell number in the spinal nucleus of the bulbocavernosus (SNB) of rats was the first neural sex difference shown to differentiate under the control of androgens, acting via classical intracellular androgen receptors. SNB motoneurons reside in the lumbar spinal cord and innervate striated muscles involved in copulation, including the bulbocavernosus (BC) and levator ani (LA). SNB cells are much larger and more numerous in males than in females, and the BC/LA target muscles are reduced or absent in females. The relative simplicity of this neuromuscular system has allowed for considerable progress in pinpointing sites of hormone action, and identifying the cellular bases for androgenic effects. It is now clear that androgens act at virtually every level of the SNB system, in development and throughout adult life. In this review we focus on effects of androgens on developmental cell death of SNB motoneurons and BC/LA muscles; the establishment and maintenance of SNB motoneuron soma size and dendritic length; BC/LA muscle morphology and physiology; and behaviors controlled by the SNB system. We also describe new data on neurotherapeutic effects of androgens on SNB motoneurons after injury in adulthood.     

Interaction of BDNF and testosterone in the regulation of adult perineal motoneurons

In androgen-sensitive motoneurons of the spinal nucleus of the bulbocavernosus (SNB), we investigated the interaction of BDNF (brain-derived neurotrophic factor) and testosterone to understand whether each factor gates the ability of the other to regulate androgen receptor expression and soma size, and whether each factor requires the presence of the other for its action. We axotomized SNB motoneurons and applied BDNF or PBS (phosphate-buffered saline) to the cut ends of the axons in rats that were castrated and treated with either testosterone or placebo. Control groups were either not castrated or not axotomized, or had intact SNB axons and were castrated and treated with testosterone or placebo. We found that testosterone determined the expression of nuclear androgen receptor, and this effect was enhanced by both BDNF and contact with the target muscles. The effect of BDNF on androgen receptor expression was seen only when testosterone was present. In the regulation of soma size, BDNF dominated. The application of BDNF completely compensated for the loss of testosterone in castrated males so that the testosterone effect on soma size was seen only in intact SNB motoneurons and in axotomized motoneurons treated with PBS. Moreover, testosterone increased androgen receptor and soma size in axotomized SNB motoneurons, indicating that testosterone can act on sites other than the target muscles of the SNB to regulate each of these. These results indicate that the regulation of androgen receptor by testosterone does not require BDNF, but the regulation of androgen receptor by BDNF does require testosterone. The regulation of soma size by BDNF does not require high expression of nuclear androgen receptor.     

Differential effects of testosterone metabolites upon the size of sexually dimorphic motoneurons in adulthood.

This study examined the effect of testosterone and two of its metabolites on the size of motoneurons in the sexually dimorphic spinal nucleus of the bulbocavernosus (SNB) in adult male rats. Treatment of castrates with either testosterone or dihydrotestosterone maintained SNB cell size, although testosterone was more effective in this regard. However, estradiol, either alone or in conjunction with dihydrotestosterone treatment, had no effect on the size of the somata or nuclei of SNB motoneurons. These results indicate that testosterone affects SNB cell size by interacting with androgen receptors and that aromatized metabolites of testosterone are not involved in this aspect of motoneuronal plasticity in adulthood. Because the penile reflexes mediated by the SNB neuromuscular system are also sensitive to androgen but not estrogen treatment, morphological changes in SNB cells may contribute to the androgenic modulation of these reflexes.     

Androgen-sensitivity of somata and dendrites of spinal nucleus of the bulbocavernosus (SNB) motoneurons in male C57BL6J mice

In rats, androgens in adulthood regulate the morphology of motoneurons in the spinal nucleus of the bulbocavernosus (SNB), including the size of their somata and the length of their dendrites. There are conflicting reports about whether androgens exert similar influences on SNB motoneurons in mice. We castrated or sham-operated C57BL6J mice at 90 days of age and, thirty days later, injected cholera toxin conjugated horseradish peroxidase into the bulbocavernosus muscle (to label SNB motoneurons) on one side, and into intrinsic foot muscles contralaterally (to label motoneurons of the retrodorsolateral nucleus (RDLN)). Castrated mice had significantly smaller SNB somas compared to sham-operated mice while there were no differences in soma size of RDLN motoneurons. Dendritic length in C57BL6J mice, estimated in 3-dimensions, also decreased significantly after adult castration. In rats, androgens act directly through androgen receptors (AR) in SNB motoneurons to control soma size and nearly all SNB motoneurons contain AR. Since SNB somata in C57BL6J mice shrank after adult castration, we used immunocytochemistry to characterize AR expression in SNB cells as well as motoneurons in the RDLN and dorsolateral nucleus (DLN). A pattern of labeling matched that seen previously in rats: the highest percentage of AR-immunoreactive motoneurons are in the SNB (98%), the lowest in the RDLN (25%) and an intermediate number in the DLN (78%). This pattern of AR labeling is consistent with the possibility that androgens also act directly on SNB motoneurons in mice to regulate soma size in mice.