Spinal cord injury induced arrest in estrous cycle of rats is ameliorated by S-nitrosoglutathione: novel therapeutic agent to treat amenorrhea

Abstract

Introduction: Amenorrhea following spinal cord injury (SCI) has been well documented. There has been little research on the underlying molecular mechanisms and therapeutics.

Aim: The purpose of the present study was to investigate the effect of GSNO in ameliorating SCI-induced amenorrhea through affecting the expression of CX43, NFkB, and ERβ protein.

Methods: SCI was induced in female SD rats at the T9-T10 level. Estrous stage was determined by vaginal smear. GSNO (50 µg/kg body weight) was gavage fed daily. Animals were sacrificed on day 7 and 14 post SCI. Ovaries were fixed for histological and biochemical studies. Expression levels of ERβ, CX-43, and NFkB were analyzed by Western blot and immunofluorescence.

Main outcome measures: GSNO hastens resumption of the estrous cycle following SCI-induced transient arrest.

Results: Resumption of estrous cycle was hastened by GSNO. Atretic and degenerating follicles seen in the ovary of SCI rats on day 14 post-SCI were decreased in GSNO treated animals. The increased CX43 expression observed with SCI ovary was decreased by GSNO. ERβ expression decreased significantly on day 7 and 14 post-SCI and was restored with GSNO treatment. Following SCI, NFkB expression was increased in the ovarian follicles and the expression was reduced with GSNO administration. The number of terminal deoxynucleotidyl transferase-mediated biotinylated uridine triphosphate (UTP) nick end labeling positive follicular and luteal cells was increased after SCI. GSNO-treated animals had significantly fewer apoptotic cells in the ovary.

Conclusion: SCI-induced amenorrhea is accompanied by an increase in CX43 expression and a decrease in ERβ expression. SCI animals treated with GSNO resumed the estrous cycle significantly earlier. These results indicate a potential therapeutic value for GSNO in treating amenorrhea among SCI patients.

Figure 1

GSNO augments the recovery from transient arrest in estrous cycle induced by SCI. Rats after SCI were divided into two groups. One received GSNO at a daily dose of 0.05 mg/kg body weight and the other saline. Estrous cycle was determined by vaginal smear method as described in material and methods. There was a significant difference in the recovery of estrus cycle between vehicle and GSNO group. Vehicle group acquired regular estrous cycle after 21 ± 1.41 days, while GSNO group in 12.83 ± 1.35 days (N: vehicle = 6; GSNO = 7; **P < 0.001 vs. Vehicle). Estrus cycle of sham operated animals (N = 4) was not affected and the animals showed regular 4 day cycle (data not shown).

Figure 2

GSNO mediated structural changes in the ovary of SCI rats. Ovary of sham (A, D), vehicle (B, E) and GSNO (C, F) treated SCI animals were observed 14 days after injury following H&E staining. Sham animals showed degeneration of corpus luteum leading to the formation of corpus albicans (A). Vehicle treated ovary of SCI rats showed defective corpus albicans formation in addition to tissue sparing (B). Ovary of GSNO treated SCI rats show no tissue degeneration and the cellular architecture resembles that of sham animals (C). Sham animals also showed many ovarian follicles in the secondary follicle stage (D). Atrophying secondary follicles were evident with ovary of vehicle treated SCI animals (E). GSNO treated SCI animals showed normal healthy secondary follicles (F). Each photograph is a representative of N = at least 4 in each group.

Figure 3

GSNO is effective in inhibiting SCI induced cellular apoptosis in the ovary of rats. (A) TUNEL-positive cells in the ovarian follicle of sham operated and vehicle and GSNO treated SCI rats. Increased number of TUNEL-positive cells was evidenced in vehicle group. Sham and GSNO group showed no apoptotic follicular cells. Lower panel shows the DAPI stained respective sections of the ovary. (B) TUNEL-positive cells in the corpus luteum of sham, vehicle and GSNO group of rats. Corpus luteum of vehicle group showed higher incidence of apoptosis when compared to sham and GSNO group. Lower panel shows the DAPI stained respective sections of the ovary. (C) The number of TUNEL-positive in the corpus luteum were quantified blindly in at least 10 different microscopic fields (N = 4 in each group) and averaged. Data are represented as mean ± SD. P < 0.001 as compared with sham.

Figure 4

Nuclear translocation of NFkB in the ovary of SCI rats. Ovary of sham and SCI rats treated with vehicle and GSNO for 14 days was immunolabelled with anti NFkB antibody. In sham operated animals, the NFkB was compartmentalized to cytoplasm (A&B). SCI and ensuing transient arrest in estrous cycle induced nuclear translocation of NFkB (C&D). Nuclear translocation of SCI induced NFkB in the ovary was inhibited by GSNO treatment (E&F). Each photograph is a representative of N = at least 4 in each group. (A, C, E; 200×: B, D, F; 400×).

Figure 5

GSNO induced down regulation of CX-43 expression in ovary of SCI rats. SCI animals were divided into two groups. One group received GSNO (0.05 mg/kg body wt); the other received the same quantity of saline. Sham animals also received saline. (A) Ovary of sham, vehicle and GSNO group or SCI rats stained with CX-43. Ovary of different groups extracted 14 days after SCI was immunostained following the routine procedure. Bottom row shows the DAPI staining of the respective sections. Increased level of CX-43 expression was seen with the ovary of vehicle treated SCI rats. Administration of GSNO at a dose of 0.05 mg/kg greatly decreased in CX-43 expression. (B) Western blots showing the expression pattern of CX-43 in the ovary of SCI rats treated with vehicle and GSNO. Tissues extracted on day 7 and 14 after SCI were resolved on 4–20% SDS PAGE and probed with anti CX-43 antibody. Loading volume was determined by B-actin expression level. CX-43 protein expression was up regulated both on day 7 and 14 in vehicle group. GSNO treatment reversed the increase in CX-43 up regulation seen with ovary of SCI rats (N: vehicle = 6; GSNO = 7). (C) Densitometry analysis of autoradiographs from at least 3 animals in each group. Data are represented as percentage over sham. ***P < 0.001.

Figure 6

ERβ expression in ovaries of SCI rats. SCI animals were grouped into two. One group received GSNO (0.05 mg/kg body wt); the other and the sham animals received saline. (A) Ovary of sham, vehicle, and GSNO group or SCI rats stained with anti ERβ antibody. Ovary of different groups extracted 14 days after SCI was immunostained following the routine procedure. Decreased level of ERβ expression was seen with the ovary of vehicle treated SCI rats. Administration of GSNO at a dose of 0.05 mg/kg greatly increased in ERβ expression. (B) Western blots showing the expression pattern of ERβ in the ovary of SCI rats treated with vehicle and GSNO. Tissues extracted on day 7 and 14 after SCI were resolved on 4–20% SDS-PAGE and probed with anti ERβ antibody. Loading volume was determined by B-actin expression level. ERβ protein expression was down regulated both on day 7 and 14 in vehicle group. GSNO treatment reversed the decrease in ERβ down regulation seen with ovary of SCI rats (N: vehicle = 6; GSNO = 7). (C) Densitometry analysis of autoradiographs from at least three animals in each group. Data are represented as percentage over sham. *P < 0.05, ***P < 0.001.