Nitric Oxide Synthase Is Involved in Follicular Development via the PI3K/AKT/FoxO3a Pathway in Neonatal and Immature Rats

Abstract

It is assumed that nitric oxide synthase and nitric oxide are involved in the regulation of female reproduction. This study aimed to assess the roles of nitric oxide synthase (NOS) in follicular development. The endothelial NOS (eNOS) inhibitor L-NAME, inducible NOS (iNOS) inhibitor S-Methylisothiourea (SMT) and NOS substrate L-arginine (L-Arg) were used in the NOS inhibition models in vivo. Neonatal female rats were treated with phosphate buffer saline (PBS, control), L-NAME (L-NG-Nitroarginine Methyl Ester, 40 mg/kg), SMT (S-Methylisothiourea, 10 mg/kg), L-NAME + SMT, or L-Arg (L-arginine, 50 mg/kg) via subcutaneous (SC) injection on a daily basis for 19 consecutive days, with the samples being collected on specific postnatal days (PD5, PD10, and PD19). The results indicated that the number of antral follicles, the activity of total-NOS, iNOS, neuronal NOS (nNOS), and eNOS, and the content of NO in the ovary were significantly (p < 0.05) increased in the L-Arg group at PD19, while those in L + S group were significantly (p < 0.05) decreased. Meanwhile, the ovarian expression in the L-Arg group in terms of p-AKT, p-FoxO3a, and LC3-II on PD19 were significantly (p < 0.05) upregulated, while the expressions of PTEN and cleaved Caspase-3 were (p < 0.05) downregulated as a result of NOS/NO generation, respectively. Therefore, the results suggest that NOS is possibly involved in the maturation of follicular development to puberty via the PI3K/ AKT/FoxO3a pathway, through follicular autophagia and apoptosis mechanisms.

Keywords: AKT; FoxO3a; PTEN; follicular development; nitric oxide synthase.

Figure 1

Effect of nitric oxide synthase (NOS) inhibitors and substrate on follicular development in neonatal and immature rats. Animals in the control, L-NG-Nitroarginine Methyl Ester (L-NAME), S-Methylisothiourea (SMT), L-arginine (L-Arg), or L + S (L-NAME plus SMT) groups were subcutaneously hypodermically injected with phosphate buffer saline (PBS), L-NAME (40 mg/kg), SMT (10 mg/kg), L-NG-Nitroarginine Methyl Ester (40 mg/kg) and S-Methylisothiourea (10 mg/kg), or L-arginine (50 mg/kg) for five (PD5, A), 10 (PD10, B), or 19 days (PD19, C) from the first day of birth (PD1). UF, unassembled follicle; PmF, primordial follicle; PF, primary follicle; SF, secondary follicle; AF, antral follicle. Bars with different letters denote significant differences (p < 0.05).

Figure 2

Effect of NOS inhibitors and substrate on ovarian NOS (total NOS, A; iNOS, B; nNOS, C; eNOS, D) activity and NO concentration (E) in neonatal and immature rats. Animals in the control, L-NAME, SMT, L-Arg or L + S (L-NAME plus SMT) groups were subcutaneously hypodermically injected with PBS, L-NG-Nitroarginine Methyl Ester (40 mg/kg), S-Methylisothiourea (10 mg/kg), or L-NG-Nitroarginine Methyl Ester (40 mg/kg) and S-Methylisothiourea (10 mg/kg), L-arginine (50 mg/kg) for five (PD5), 10 (PD10), or 19 days (PD19) from the first day of birth (PD1). Bars with different letters denote significant differences (p < 0.05).

Figure 3

Effect of NOS inhibitors and substrate on ovarian expression of PTEN (A), p-AKT (B), AKT (C), p-FoxO3a (D), and FoxO3a (E) in neonatal rats. Animals in the control, L-NAME, SMT, L-Arg, or L + S (L-NAME plus SMT) groups were subcutaneously hypodermically injected with PBS, L-NG-Nitroarginine Methyl Ester (40 mg/kg), S-Methylisothiourea (10 mg/kg), L-NG-Nitroarginine Methyl Ester (40 mg/kg) and S-Methylisothiourea (10 mg/kg), or L-arginine (50 mg/kg) for five days (PD5) from the first day of birth (PD1). β-tubulin was used as an internal control, wherein the signal intensity was plotted as the ratio of target protein to β-tubulin. Bars with different letters denote significant differences (p < 0.05).

Figure 4

Effect of NOS inhibitors and substrate on ovarian expression of PTEN (A), p-AKT (B), AKT (C), p-FoxO3a (D), and FoxO3a (E) in immature rats. Animals in the control, L-NAME, SMT, L-Arg, or L + S (L-NAME plus SMT) groups were subcutaneously hypodermically injected with PBS, L-NG-Nitroarginine Methyl Ester (40 mg/kg), S-Methylisothiourea (10 mg/kg), L-NG-Nitroarginine Methyl Ester (40 mg/kg) and S-Methylisothiourea (10 mg/kg), or L-arginine (50 mg/kg) for 10 days (PD10) from the first day of birth (PD1). As an internal control, β-tubulin was used, wherein the signal intensity was plotted as the ratio of target protein to β-tubulin. Bars with different letters denote significant differences (p < 0.05).

Figure 5

Effect of NOS inhibitors and substrate on ovarian expression of PTEN (A), p-AKT (B), AKT (C), p-FoxO3a (D), and FoxO3a (E) in immature rats. Animals in the control, L-NAME, SMT, L-Arg, or L + S (L-NAME plus SMT) groups were subcutaneously hypodermically injected with PBS, L-NG-Nitroarginine Methyl Ester (40 mg/kg), S-Methylisothiourea (10 mg/kg), L-NG-Nitroarginine Methyl Ester (40 mg/kg) and S-Methylisothiourea (10 mg/kg), or L-arginine (50 mg/kg) for 19 days (PD19) from the first day of birth (PD1). As an internal control, β-tubulin was used, wherein the signal intensity was plotted as the ratio of target protein to β-tubulin. Bars with different letters denote significant differences (p < 0.05).

Figure 6

Effect of NOS inhibitors and substrate on ovarian expression of LC3-II (A,C,E) and cleaved-Caspase-3 (B,D,F) in neonatal and immature rats. Animals in the control, L-NAME, SMT, L-Arg, or L + S (L-NAME plus SMT) groups were subcutaneously hypodermically injected with PBS, L-NG-Nitroarginine Methyl Ester (40 mg/kg), S-Methylisothiourea (10 mg/kg), L-NG-Nitroarginine Methyl Ester (40 mg/kg) and S-Methylisothiourea (10 mg/kg), or L-arginine (50 mg/kg) for five (PD5), 10 (PD10) and 19 days (PD19) from the first day of birth (PD1). As an internal control, β-tubulin was used, wherein the signal intensity was plotted as the ratio of target protein to β-tubulin. Bars with different letters denote significant differences (p < 0.05).