A: The weight of seminal vesicles was measured after removing the adhering tissue and fluid. Wild type (WT) mice and SOD1-G93A (SOD1) mice were implanted with either a DHT-filled or an empty silastic tube. Orchidectomy and/or silastic tube implant was performed at postnatal day 75 (P75), and the seminal vesicle weight was measured at P120. DHT-treated WT mice showed a 19% increase in seminal vesicle weight (156±10.6 mg, p = 0.042) compared with control WT mice (132.1±6.4 mg). In SOD1 mice, the DHT-filled silastic tube also increased the seminal vesicle weight by 38% (146.1±5.5 mg, p = 0.0003) compared with control SOD1 mice (105.8±4.6 mg). Conversely, orchidectomy decreased the seminal vesicle weight in both WT mice (28.4±13 mg, p<0.0001) and SOD1 mice (20.8±3.5 mg, p<0.0001) compared with control WT and SOD1 mice, respectively. SOD1 control mice showed 20% reduced seminal vesicle weight compared with WT control mice (p = 0.0039). Sample size is indicated in ( ) for each group. Data are mean ± SEM. # p<0.05, ### p<0.001 (compared with age-matched WT mice), ** p<0.01, ***p<0.001 (compared with control SOD1 mice). B: WT and SOD1 mice were implanted with either a DHT-filled or an empty silastic tube at P75, and the seminal vesicles were obtained at P120. Representative pictures of the seminal vesicles are shown. Scale bar = 5 mm.

WT and SOD1 mice were implanted with either a DHT-filled or an empty silastic tube, or orchidectomized at P75, and the morphological analyses were made at P120. A: In WT mice, DHT increased the GN weight by 7% (212.4±8.8 mg), whereas orchidectomy decreased it by 14% (171.4±9.0 mg) compared with control WT mice (198.4±5.5 mg). In SOD1 mice, DHT increased the GN weight by 32% (147.5±4.5 mg, p = 0.00017), whereas orchidectomy decreased it by 25% (83.4±5.7 mg, p = 0.0086) compared with control SOD1 mice (111.4±6.6 mg). B: In WT mice, DHT increased the weight of TA muscle by 12% (71.2±2.5 mg), whereas orchidectomy decreased it by 12% (56.1±3.6 mg) compared with control WT mice (63.6±1.5 mg). In SOD1 mice, DHT increased the weight of TA muscle by 43% (46.3±2.4 mg, p = 0.0017), whereas orchidectomy decreased it by 22% (25.2±2.4 mg, p = 0.05) compared with control SOD1 mice (32.5±2.5 mg). Sample size is indicated in ( ) for each group. ## p<0.01, ### p<0.01 (compared with age-matched WT mice), ** p<0.01, ***p<0.001 (compared with control SOD1 mice). C: DHT increased the cross-sectional area of TA muscle by 22.3% (3.23±0.19 mm², n = 3, p = 0.034), whereas orchidectomy decreased it by 20.8% (2.09±0.11 mm², n = 3, p = 0.008) compared with control SOD1 mice (2.64±0.03 mm², n = 3). D: DHT did not cause a significant increase in the muscle fiber number (4.8% increase, 3020.7±152.2, p = 0.49). Likewise, orchidectomy did not cause a significant decrease in the muscle fiber number (7.0% decrease, 2681.0±86.9, p = 0.19) compared with control SOD1 mice (2883.7±97.0). E: Representative pictures of the cross sectional area of TA muscles are shown. Scale bar = 50 µm. F: Distribution of the area of single muscle fiber is shown. Per TA muscle, 600–900 muscle fibers were measured, and 3 TA muscles per each treatment group were used for the analysis of muscle fiber area. DHT treatment shifted the area of muscle fibers toward larger areas (1070.8±39.8 µm², p = 0.032), whereas orchidectomy shifted it toward smaller areas (729.1±61.9 µm², p = 0.023) compared with control SOD1 mice (904.7±26.6 µm²). Data are mean ± SEM. p<0.001 (2 way-ANOVA).

A: DHT-treated SOD1 mice showed heavier body weight compared with control SOD1 mice (p<0.001), although it was still lower than the weight of WT mice throughout all time points. Orchidectomized SOD1 mice demonstrated reduced body weights compared with control SOD1 mice (p = 0.045). Data are mean ± SEM. B: The grip-strength meter was used to assess the muscle strength, and the maximum tension generated by the grip of a mouse on the pull bar was recorded. SOD1 mice exhibited diminished grip-strength compared with WT mice throughout all time points examined (p<0.0001). DHT-treated SOD1 mice showed stronger grip-strengths compared with control SOD1 mice (p<0.001), and the gap between these two groups gradually increased as age advanced. Data are mean ± SEM.

A: The TA muscles were collected from DHT-treated SOD1 and control SOD1 mice at P120 to check the expression of insulin-like growth factor (IGF) -1 and IGF-2 through quantitative RT-PCR. DHT-treated SOD1 mice showed increased expression of IGF-1 and IGF-2, by approximately 4-fold (p = 0.0261), and 2-fold (p = 0.015), respectively, compared with control SOD1 mice. *p<0.05. B: By using quantitative RT-PCR, we found that DHT-treated SOD1 mice showed a trend of decreased expression of MuRF-1 by 44% compared with control SOD1 mice (p = 0.198).

SOD1/YFP double transgenic mice expressing yellow fluorescence protein (YFP) in all motor nerves were implanted with either a DHT-filled or an empty silastic tube, or orchidectomized at P75, and the TA muscle and the diaphragm (DIA) muscle were collected at P120, and stained with anti-α-bungarotoxin to label post-synaptic acetylcholine receptor (AChR) clusters. A: When a pre-synaptic nerve terminal (in green) fully overlaps with the post-synaptic AChR clusters (in red), the neuromuscular junction (NMJ) is defined as a “fully innervated NMJ” (d). However, if a nerve terminal is partially overlapped with AChR, or is completely absent, leaving only AChR, the NMJ is defined as a “partially innervated NMJ” (e) or a “denervated NMJ” (f), respectively. DHT-treated SOD1/YFP mice (b) showed improved NMJ innervation in the TA muscle compared with control SOD1/YFP mice (a). However, orchidectomy in SOD1/YFP mice aggravated denervation at NMJs (c). B: Quantification of NMJs at P120 in the TA muscle of DHT-treated, control, and orchidectomized SOD1/YFP mice is shown. Compared to control SOD1/YFP mice, which showed 22.3±5.7% of fully innervated NMJs, DHT-treated SOD1/YFP mice showed 47.3±14.1% of fully innervated NMJs. Orchidectomized SOD1/YFP mice showed only 9.6±3.4% of fully innervated NMJs. C: Quantification of NMJs at P120 in the DIA muscle of DHT-treated, and control SOD1/YFP mice is shown. Compared to SOD1/YFP mice, which showed 65.2±5.6% of fully innervated NMJs, DHT-treated SOD1/YFP mice showed 81.0±5.5% of fully innervated NMJs. Data are mean ± SEM. *p<0.05.

A: The phrenic nerve at the entry of the diaphragm muscle was sectioned to observe myelinated axons at P120. Representative pictures of the phrenic nerve from WT, control SOD1, and DHT-treated SOD1 mice are shown. B: Quantification of myelinated axon number in the phrenic nerves is shown. The number of myelinated axons in the phrenic nerve of SOD1 mice (184.6±10.9) is about 40% less compared with WT mice (313.0±11.5, p = 0.0003). DHT-treated SOD1 mice showed 26% more myelinated axons (232.3±16.8, p = 0.043) compared with control SOD1 mice. Sample size is indicated in ( ) for each group. *p<0.05 (compared with control SOD1 mice), ### p<0.001 (compared with WT mice). C: Representative cross-sectional pictures of the ventral roots of the spinal cord lumbar 4 segment from WT, control SOD1, DHT-treated SOD1, and orchidectomized SOD1 mice at P120 are shown. D: Quantification of myelinated axon number in the ventral root of the lumbar 4 spinal cord is shown. The total number of myelinated axons in control SOD1 mice (691.2±43.6) is about 30% less compared with that in WT mice (996.5±58.5, p = 0.0012). DHT-treated SOD1 showed 24% more total myelinated axon number compared with control SOD1 mice (859.6±53.4, p = 0.013). Especially, the number of the large caliber axons (≥4 µm) were 43% less in SOD1 mice (404.2±31.1) compared with WT mice (706.5±51.6, p = 0.0005). DHT-treated SOD1 mice showed 36% more large caliber axons (≥4 µm) (547.8±22.2, p = 0.006) compared with control SOD1 mice. Orchidectomized SOD1 mice showed 10% less total myelinated axon number compared with control SOD1 mice (622.3±29.6, p = 0.01). Sample size is indicated in ( ) for each group. Data are mean ± SEM. *p<0.05, ** p<0.01 (compared with the total myelinated axon numbers in SOD1 control), ##p<0.01, ### p<0.001 (compared with the number of large caliber axons in SOD1 control).

A: SOD1 mice were implanted with either a DHT-filled or an empty silastic (control) tube at P75, and the lumbar spinal cord (L) 3-L5 was collected at P120, the symptomatic age. Cross-sectional pictures of the hemi-lumbar spinal cord in WT, control SOD1, and DHT-treated SOD1 mice are shown. B: There was a 41% reduction in motoneuron number in the L3–L5 of SOD1 mice (13.9±0.7, n = 5) compared with WT mice at P120 (23.8±0.9, n = 4, p<0.0001). DHT-treated SOD1 mice contained 27% more motoneurons (17.7±0.8, n = 5, p = 0.0093) compared with control SOD1 mice. In the cervical spinal cord, there were 45% less motoneurons in SOD1 mice (16.8±0.7, n = 3, p = 0.015) compared with WT mice at P120 (30.6±1.5, n = 3). There were 18% more motoneurons in DHT-treated SOD1 mice (19.9±0.4, n = 3, p = 0.467) compared with control SOD1 mice. Data are mean ± SEM. **p<0.01(compared with control SOD1 mice), #p<0.05, ### p<0.001 (compared with age-matched WT mice).

A: SOD1 mice were implanted with either a DHT-filled or an empty silastic (control) tube at P75, and their motor performances were assessed every 5 days by the rota-rod test. A mouse was placed on the rotating rod at 11 rpm, and the latency until the mouse fell from the rotating rod was recorded in seconds. DHT-treated SOD1 mice (filled square, n = 16) stayed longer on the rotating rod compared with control SOD1 mice (empty circle, n = 22). Data are mean ± SEM. p = 0.043 (2-way ANOVA). B: Hindlimbs were placed into non-toxic paints and a mouse was allowed to walk on a 50 cm- length of paper, and its continuous locomotion was recorded. Representative footprints obtained from DHT-implanted or empty tube-implanted SOD1 mice at P100 are shown. DHT-treated SOD1 mouse (top panel) showed a longer step length without a trace of dragging hindlimb compared with control SOD1 mice (bottom panel). C: Step length was quantified by dividing walking distance by the number of the steps taken within the measured walking distance. DHT-treated SOD1 mice (filled square, n = 6) showed longer step length compared with control SOD1 mice (empty circle, n = 7). p = 0.003 (paired t-test). D: DHT-treated SOD1 mice showed significantly increased survival duration after DHT administration by 11% in DHT-treated SOD1-G93A mice (82.9±2.9 days, n = 18) compared with age-matched control SOD1-G93A mice (74.8±2.2 days, n = 24). E: DHT-treated SOD1 mice showed significantly increased the overall lifespan by 8 days (filled square, 157.9±2.9 days, n = 18) compared with control SOD1 mice (empty circle, 149.8±2.2 days, n = 24). p = 0.0174 (log-rank test).