行政院國家科學委員會補助專題研究計畫成果報告
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※ 肌肉雄性素敏感度對運動神經元之影響 ※
※ Influence of androgen sensitivity of target muscles on ※
※ innervating motoneurons ※
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計畫類別：X□個別型計畫 □整合型計畫 計畫編號：NSC 89－2320 － B －038 － 077 － 執行期間： 89 年 11 月 01 日至 90 年 07 月 31 日
計畫主持人：楊良友 共同主持人：
本成果報告包括以下應繳交之附件：
□赴國外出差或研習心得報告一份
□赴大陸地區出差或研習心得報告一份
□出席國際學術會議心得報告及發表之論文各一份
□國際合作研究計畫國外研究報告書一份
執行單位：台北醫學大學生理學科
中 華 民 國 90 年 10 月 29 日
行政院國家科學委員會專題研究計畫成果報告
肌肉雄性素敏感度對運動神經元之影響
Influence of andr ogen sensitivity of tar get muscles on inner vating motoneur ons
計畫編號：NSC 89-2320-B-038-077
執行期限：89 年 11 月 01 日至 90 年 07 月 31 日 主持人：楊良友 台北醫學大學生理學科
一、中文摘要
SNB 運動神經元展現高度的雄性素受體而其所控制 的 BC/ LA 肌肉大小隨體內雄性素含量的多寡而改 變。相對地，GP 運動神經元展現少量的雄性素受 體而其所控制的 GP 肌肉大小不隨體內雄性素含量 的變化而改變。這些觀察引出了一個非常重要的問 題，那就是肌肉對雄性素的敏感度是否決定運動神 經元雄性素受體的展現。在本計劃中，我們以手術 操縱的方式來探討上述的問題。在同一隻老鼠身 上，一側讓雄性素敏感的運動神經元控制非雄性素 敏感的肌肉，另外一側則維持以雄性素敏感的運動 神經元控制雄性素敏感的肌肉。同樣地，對另一組 老鼠，我們也以手術操縱，讓非雄性素敏感的運動 神經元控制雄性素敏感的肌肉。手術後四十天，我 們在肌肉注射 CT-HRP 並於二天後將動物犧牲。我 們的結果發現絕大部分被 HRP 所染到的 SNB 運動神 經元展現中高度的雄性素受體免疫化學反應，然而 卻只有一小部的 GP 運動神經元展現中高度的雄性 素受體免疫化學反應。除此之外，SNB 細胞核大小 及其所控制的肌肉重量在手術後重新連接控制 BC/LA 肌肉與手術後被迫連接控制 GP 肌肉兩者之 間並無差異。再者，SNB 運動神經元被迫連接控制 GP 肌肉及 GP 運動神經元被迫連接控制 BC/LA 肌 肉，其中高度雄性素受體的免疫化學反應並沒有加 強或減弱。以上結果並不支持運動神經元的雄性素 受體展現受其所控制肌肉之雄性素敏感度所影響。
關鍵詞：雄性素敏感的運動神經元，SNB 運動神經 元，GP 運動神經元，BC/LA 肌肉，GP 肌肉，雄性素 受體，雄性素
Abstract
Motoneurons in the spinal nucleus of the bulbocavernosus (SNB) express high levels of androgen receptors and their target bulbocavernosus and levator ani (BC/LA) muscle is androgen-sensitive. In contrast, gracilis posticus (GP) motoneurons exhibit low levels of androgen receptors and their target muscle is androgen-insensitive. The above observations lead us to question whether the androgen sensitivity of target muscle determines or induces the androgen receptor expression of innervating motoneurons. In this study, we tested the hypothesis that androgen receptor expression of motoneurons is determined by the androgen sensitivity of target muscle by cross-innervating motoneurons with high (SNB) and low (GP) levels of androgen receptors, respectively, with androgen-insensitive GP muscle and androgen-sensitive BC/LA muscle. Forty days following surgery, we injected CT-HRP into the target muscle and examined the HRP labeling and androgen receptor expression in these SNB and GP motoneurons 2 days after HRP injection. Our results indicate that the majority of SNB motoneurons retrogradely-labeled by HRP show medium-high AR-LI, whereas only a small
portion of GP motoneurons
retrogradely-labeled by HRP express medium-high AR-LI. In addition, the nuclear size and the weight of muscle innervated by SNB motoneurons on the side reinnervating BC/LA muscle are similar to those on the side cross-innervating GP
muscle. Furthermore, cross-innervation of SNB and GP motoneurons, respectively, with GP muscle and BC/LA muscle did not decrease or increase the percentage of retrogradely labeled neurons with medium-high androgen receptor-like immunoreactivity (AR-LI). These results strongly suggest that the androgen sensitivity of the target muscle does not determine the androgen receptor expression of innervating motoneurons.
Keywords: Androgen-sensitive motoneurons, SNB motoneurons, GP
motoneurons, BC/LA muscle, GP muscle, Androgen receptor, Androgens
Introduction
The motoneurons in the spinal nucleus of the bulbocavernosus (SNB) and their target bulbocavernosus and levator ani (BC/LA) muscle are sexually dimorphic and both are androgen-dependent in rats (Arnold 1984;Arnold et al. 1988;Arnold and Jordan 1988;Breedlove and Arnold 1980;Breedlove and Arnold 1981;Jordan et al. 1997).
Male rats have larger and more SNB motoneurons as well as larger BC/LA muscle than females (Arnold, Matsumoto, and Micevych 1988;Arnold and Jordan 1988;Breedlove and Arnold 1980). Both SNB motoneurons and BC/LA muscle express high levels of androgen receptors (Arnold, Matsumoto, and Micevych 1988;Arnold and Jordan 1988;Jordan, Padgett, Hershey, Prins, and Arnold 1997;Matsumoto et al. 1996;Yang and Arnold 2000b;Yang and Arnold 2000a). In contrast, the gracilis posticus (GP) motoneurons express low levels of androgen receptors and innervates androgen-insensitive GP muscle (unpublished observation).
Innervation of androgen-sensitive muscle by motoneurons with high levels of androgen receptor raises an important issue on whether the androgen sensitivity of motoneurons determines that of their target muscle or vice versa. Testosterone influenced the soma size of SNB motoneurons re-innervating androgen-sensitive BC/LA muscle, but did not alter that of SNB motoneurons cross-innervating androgen-insensitive soleus muscle (Araki et al. 1991). Based on this finding, we hypothesize that the androgen sensitivity of the target muscle determines or controls the androgen sensitivity and androgen receptor expression of innervating motoneurons. In this study, therefore, we investigated the androgen receptor expression of androgen-sensitive motoneurons after reinnervating androgen-sensitive muscle and cross-innervating androgen-insensitive muscle. Furthermore, we also examined how the motoneurons with low levels of androgen receptor respond to the change of androgen sensitivity of target muscle.
Mater ials and Methods
Adult male Sprague-Dawley rats were used in this experiment. Animals were housed in a 12h:12h light-dark cycle and had free access to water and food all the time.
For all surgeries, animals were anesthetized by sodium pentobarbital at a dose of 50 mg/kg and supplemented by gas anesthetic, if necessary. Seventeen males were randomly assigned to three groups. In Group I (N=6), the SNB axons were cut bilaterally at the place close to the BC/LA muscle. The SNB axons were resutured to the BC/LA muscle on one side and sutured into the denervated GP muscle on the other side (SNB-BC/LA muscle and SNB-GP muscle). In Group II (N=6), the GP axons were cut bilaterally at the place close to the GP muscle. The GP
axons were then resutured to the GP muscle on one side and sutured to the denervated BC/LA muscle on the other side (GP-GP muscle and GP-BC/LA muscle). In Group III (N=5), we unilaterally cut the SNB axons and GP axons and resutured them to the BC/LA muscle and the GP muscle, respectively (SNB-BC/LA muscle and GP-GP muscle). In all surgeries of cross-innervation, the distal stump of the original nerve innervating the muscle was cauterized and ligated to prevent reinnervation. Forty days (based on empirical experience in pilot animals) after cross- or re-innervation surgery, 0.2%
CT-HRP (6 µl per side) was injected into the BC/LA or GP muscle. Animals were sacrificed 2 days later. Before sacrificing the animals, we stimulated the nerve and checked if the nerve functionally innervated the muscle. All animals were perfused transcardially with heparinized 0.9% NaCl (10 ml), 5% Acrolein (60 ml), and 2% (240 ml) paraformaldehyde in sequential order.
Spinal cords was post-fixed in 5% Acrolein for 3 hours at 4 0C and transferred to 20%
sucrose for cryoprotection until they sank.
We sectioned spinal cords longitudinally at 50 µm. Spinal sections including SNB region were processed for HRP reaction first and then androgen receptor (PG-21) immunocytochemistry (ICC). The procedures for androgen receptor-like immunoreactivity (AR-LI) were described in detail in the paper published by Yang and Arnold (Yang and Arnold 2000b;Yang and Arnold 2000a). The location of retrogradely transported HRP product, the levels of androgen receptor-like immunoreactivity (AR-LI) of SNB or GP motoneurons were measured. For the measurement of AR-LI, forty HRP-labeled SNB or GP motoneurons were selected from the 2 or 3 sections containing the majority of HRP-labeled neurons if the total number of HRP-labeled neurons exceeded 40. The AR-LI of each neuron was judged as none, low, medium or high. The percentage of HRP-labeled SNB or GP motoneurons with medium-high AR-LI was calculated and used for further analysis. If the total number of
HRP-labeled SNB or GP motoneurons was smaller than 40, the AR-LI of all the HRP-labeled SNB or GP motoneurons was judged and the percentage of medium-high AR-LI was calculated and used for further analysis.
Statistics
Comparison of data between Group I and Group II and Comparison of data between Group I and Group III were analyzed by 2x2 Repeated measures ANOVA followed by Student-Newman-Keuls’ (SNK) post hoc tests. An α level of 0.05 was used for all statistical tests.
Results:
Electrical stimulation of the motor branch of the pudendal nerve or axons of GP motoneurons resulted in contraction of the cross-innervated muscle or reinnervated muscle. In addition, injection of retrograde tracer HRP into the cross-innervated muscle or reinnervated muscle labeled the SNB motoneurons or GP motoneurons. These results proved that cross-innervation or reinnervation treatment was successful.
Our results indicated that cross-innervation of androgen-sensitive SNB motoneurons with androgen-insentitive GP muscle dramatically decreased the raw number of HRP-labeled SNB motoneurons.
In contrast, cross-innervation of GP motoneurons with androgen-sensitive BC/LA muscle did not affect the raw number of HRP-labeled GP motoneurons. Two way repeated measures ANOVA showed a significant treatment (reinnervation vs.
cross-innervation) effect [F(1, 10) =28.572, p
= 0.0003 ] and a significant group (SNB vs.
GP) X treatment (reinnervation vs.
cross-innervation) interaction [F(1, 10) = 13.909, p = 0.0039]. SNK post-hoc tests revealed that the raw number of retrogradely-labeled HRP neurons was significantly lower in SNB motoneurons cross-innervating GP muscle than in those reinnervating BC/LA muscle (p<0.01).
There was no difference in the raw number of retrogradely-labeled HRP neurons between GP motoneurons reinnervating GP muscle and those cross-innervating BC/LA muscle.
SNB motoneurons expressed higher levels of androgen receptor than GP motoneurons no matter they reinnervated BC/LA muscle or cross-innervated GP muscle. Cross-innervation of SNB motoneurons with GP muscle led to a significant reduction of HRP-labeled SNB motoneurons with medium-high AR-LI.
For the number of retrogradely-labeled HRP neurons with medium-high AR-LI, two-way repeated measures ANOVA showed a group effect [F(1, 10) = 45.256, p < 0.0001], a treatment effect [F(1, 10) = 48.101, p <
0.0001], and a group X treatment interaction [F(1, 10) = 28.215, p = 0.0003]. SNK post-hoc tests revealed that the number of retrogradely-labeled HRP neurons with medium-high AR-LI was significantly higher in SNB motoneurons than in GP motoneurons (p < 0.01). Cross-innervation of SNB motoneurons with GP muscle significantly reduced the number of retrogradely-labeled HRP neurons with medium-high AR-LI compared to those reinnervating BC/LA muscle (p < 0.01). No difference was found in this measure between GP motoneurons cross-innervating BC/LA muscle and those reinnervating GP muscle.
The majority of the HRP-labeled SNB
motoneurons expressed medium-high AR-LI, whereas only a small percentage of HRP-labeled GP motoneurons exhibited medium-high AR-LI. Cross-innervation did not increase or decrease the androgen receptor expression in HRP-labeled SNB or GP motoneurons. For the percentage of retrogradely-labeled HRP neurons with medium-high AR-LI, two-way repeated measures ANOVA revealed a significant group (SNB vs. GP) effect [F(1, 10) = 71.192, p < 0.0001]. The percentage of HRP-labeled neurons with medium-high AR-LI was significantly higher in SNB motoneurons than in GP motoneurons.
Both SNB motoneurons cross-innervating GP muscle and those reinnervating BC/LA muscle exhibited a high percentage of retrogradely-labeled HRP neurons with medium-high AR-LI and there was no difference in this measure between SNB motoneurons cross-innervating GP muscle or reinnervating BC/LA muscle. In contrast, only a small fraction of HRP-labeled GP motoneurons showed medium-high AR-LI no matter they reinnervated GP muscle or cross-innervated BC/LA muscle.
Neither cross-innervation of SNB motoneurons with GP muscle nor cross-innervation of GP motoneurons with BC/LA muscle affected the weight of androgen-sensitive BC/LA or androgen-insensitive GP muscle. Two-way repeated measures ANOVA did not show any group effect, treatment effect or group x treatment interaction in the comparison of muscle weight innervated by SNB motoneurons or GP motoneurons. No significant difference was found among BC/LA muscles and GP muscles no matter
they were innervated by SNB motoneurons or by GP motoneurons.
The nuclea size of SNB motoneurons reinnervating BC/LA muscle was significantly smaller in animals with contralateral intact SNB motoneurons than in those with contralateral cross-innervation of GP muscle. Nonetheless, the nuclear size of SNB motoneurons did not differ between SNB motoneurons reinnervating BC/LA muscle and those cross-innervating GP muscle. For comparison of the SNB nuclear size between Group I (SNB-GP/SNB-BC/LA) and Group III (Intact SNB/ SNB-BC/LA), two-way repeated measures ANOVA indicated a significant group effect (Intact
SNB/ SNB-BC/LA vs.
SNB-GP/SNB-BC/LA) [F(1, 9) = 19.089, p = 0.0018]. The nuclear size was significantly smaller in animals of Group I than in those of Group III.
Discussion
We have successfully cross-innervated SNB motoneurons and GP motoneurons with the GP muscle and BC/LA muscle, respectively, in adult male rats. This is manifested by the HRP labeling of motoneurons following injection of the retrograde tracer HRP into the cross-innervated muscle and the observed muscle contraction elicited by electrical stimulation of the motor branch of the pudendal nerve or GP axons originating from cross-innervating motoneurons.
The BC/LA muscle is
androgen-sensitive, but the GP muscle is androgen-insensitive. Numerous evidence has demonstrated that castration decreases
the BC/LA muscle weight and testosterone treatment after castration prevents or reverses the castration-induced reduction of the BC/LA muscle weight (Arnold, Matsumoto, and Micevych 1988;Arnold and Jordan 1988).
The GP muscle weight is not influenced by the testosterone levels. This is manifested by the fact that there was no difference in the GP muscle weight among castrates, intact male rats, and castrates treated with testosterone (unpublished observations).
SNB motoneurons cross-innervating the androgen-insensitive GP muscle had significantly fewer neurons labeled by the retrograde tracer HRP than those reinnervating BC/LA muscle. In contrast, no difference was found in the raw number of retrogradely-labeled HRP neurons between GP motoneurons reinnervating androgen-insensitive GP muscle and those cross-innervating androgen-sensitive BC/LA muscle.
SNB motoneurons expressed high levels of androgen receptors, whereas the majority of GP motoneurons showed low or no androgen receptor expression. Following injection of retrograde tracer HRP into the GP muscle (on the reinnervated side) or BC/LA muscle (on the cross-innervated side), only a few HRP-labeled GP motoneurons expressed medium-high AR-LI. In contrast, the majority of SNB motoneurons labeled by HRP showed medium-high AR-LI no matter they reinnervated the androgen sensitive BC/LA muscle or cross-innervated the androgen-insensitive GP muscle.
Our findings strongly suggest that the androgen sensitivity of the target muscle does not affect the androgen receptor expression of the innervating motoneurons in adult rats.
Cross-innervation of SNB motoneurons with GP muscle dramatically decreased the number of HRP-labeled SNB neurons with medium-high AR-LI but did not affect the percentage of HRP-labeled SNB neurons with medium-high AR-LI. In contrast, there was no difference in the raw number and the percentage of HRP-labeled GP neurons with medium-high AR-LI between the GP motoneurons reinnervating GP muscle and those cross-innervating BC/LA muscle.
The difference in the number of HRP-labeled SNB neurons with medium-high AR-LI between the SNB motoneurons reinnervating BC/LA muscle and those cross-innervating GP muscle may result from the difference in the shape of the BC/LA (spindle) muscle and GP muscle (long and flat), which influences the absorption rate of HRP.
The muscle weights of GP muscle and BC/LA muscle are comparable and are not influenced by cross-innervation or reinnervation manipulation. In intact adult male rats, the GP muscle weight is similar to that of BC/LA muscle (Unpublished observation). In this study, we compared the weights of GP muscle and BC/LA muscle following cross-innervation or reinnervation manipulation and we found no difference in the weight of BC/LA muscle and GP muscle after cross-innervation or reinnervation treatment. These findings exclude the possibility that the weight of target muscle may influence the androgen receptor expression of innervating motoneurons.
The nuclear size seems to have no effect on the expression of medium-high AR-LI in the SNB motoneurons. The nuclear size of SNB motoneurons cross-innervating the GP muscle is not different from that of SNB
motoneurons reinnervating the BC/LA muscle. In addition, no difference is found in the percentage of HRP-labeled neurons with medium-high AR-LI in the SNB motoneurons cross-innervating the GP muscle or reinnervating the BC/LA muscle.
These above findings together exclude the possible contribution of nuclear size to the lack of difference in the androgen receptor expression between SNB motoneurons cross-innervating the GP muscle and those reinnervating the BC/LA muscle.
Although the androgen sensitivity of target muscle has been reported to affect the soma size of innervating motoneurons (Araki, Harada, and Kuno 1991), our current results did not support the idea that the androgen sensitivity of the target muscle influences the androgen receptor expression of innervating motoneurons. Neither the weight of the target muscles nor the nuclear size contributes to the lack of difference in the androgen receptor expression between SNB motoneurons reinnervating the BC/LA muscle and those cross-innervating the GP muscle.
Refer ences
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