J Vet Sci.  2011 Sep;12(3):257-265. 10.4142/jvs.2011.12.3.257.

Influence of nitric oxide on in vitro growth, survival, steroidogenesis, and apoptosis of follicle stimulating hormone stimulated buffalo (Bubalus bubalis) preantral follicles

• Dubey PK
• Tripathi V
• Singh RP
• Sharma GT

• KMID: 1067398
• DOI: http://doi.org/10.4142/jvs.2011.12.3.257

Abstract

Effect of sodium nitroprusside (SNP), a nitric oxide (NO) donor, on in vitro survival, growth, steroidogenesis, and apoptosis of buffalo preantral follicles (PFs) was investigated. PFs (200~250 microm) were isolated by micro-dissection and cultured in 0 (control), 10(-3), 10(-5), 10(-7), and 10(-9) M SNP. To examine the reversible effect of SNP, PFs were cultured with 10(-5) M SNP + 1 mM Nomega-nitro-L-arginine methyl ester (L-NAME) or 1.0 microg hemoglobin (Hb). The results showed that greater concentrations of SNP (10(-3), 10(-5), 10(-7) M) inhibited (p < 0.05) FSH-induced survival, growth, antrum formation, estradiol production, and oocyte apoptosis in a dose-dependent manner. However, a lower dose of SNP (10(-9) M) significantly stimulated (p < 0.05) the survival, growth, antrum formation, follicular oocyte maturation, and stimulated progesterone secretion compared to the control. A combination of SNP + L-NAME promoted the inhibitor effect of SNP while a SNP + Hb combination reversed this effect. Nitrate and nitrite concentrations in the culture medium increased (p < 0.05) in a dose-dependent manner according to SNP concentration in the culture medium. At higher concentrations, SNP had a cytotoxic effect leading to follicular oocyte apoptosis whereas lower concentrations have stimulatory effects. In conclusion, NO exerts a dual effect on its development of buffalo PFs depending on the concentration in the culture medium.

Keyword

apoptosis; buffalo; nitric oxide; preantral follicle; steroidogenesis

MeSH Terms

Animals
*Apoptosis
Buffaloes/*physiology
Estradiol/biosynthesis
Female
Follicle Stimulating Hormone/metabolism
NG-Nitroarginine Methyl Ester/pharmacology
Nitrates/pharmacology
Nitric Oxide/*metabolism
Nitric Oxide Donors/pharmacology
Nitrites/pharmacology
Nitroprusside/pharmacology
Oocytes/cytology/drug effects/growth & development/metabolism
Ovarian Follicle/*cytology/drug effects/growth & development/*metabolism
Progesterone/biosynthesis

Figure

• 

  Fig. 1 Effect of SNP (0, 10-9, 10-7, 10-5, and 10-3 M) with or without L-NAME (1.0 mM) and Hb (1.0 µg) on the accumulation of estradiol in the buffalo PF culture medium during different culturing days. Lines with different superscript letters differ significantly (p < 0.05) when comparing different treatments on the same day. SNP: sodium nitroprusside, L-NAME: Nω-nitro-L-arginine methyl ester, Hb: hemoglobin. Each value represents the means ± SE of a total of three measurement from five independent experiments.

• 

  Fig. 2 Effect of SNP (0, 10-9, 10-7, 10-5, and 10-3 M) with or without L-NAME (1.0 mM) and Hb (1.0 µg) on the accumulation of progesterone in the PF culture media on different culturing days. Lines with different superscript letters differ significantly (p < 0.05) when comparing different treatments on the same day. Each value represents the means ± SE of a total of three measurements from five independent experiments.

• 

  Fig. 3 Effect of SNP (0, 10-9, 10-7, 10-5, and 10-3 M) with or without L-NAME (1.0 mM) and Hb (1.0 µg/mL) on NO-3/NO-2 concentrations in the PF culture medium on different culturing days. Values with different superscripts letters differ significantly (p < 0.05) when comparing different treatments on the same day. Each value represents the means ± SE of a total of three measurement from five independents experiments.

• 

  Fig. 4 In vitro development of buffalo PFs cultured with SNP (10-9 M). (A) PFs showing centrally located oocyte surrounded by layers of granulosa cells on day 0. (B) In vitro growth of PFs after 6 days of culturing. (C) PFs showing signs of antrum formation (a) after 8 days of culturing. (D) Initiation of antrum formation after 10 days of culturing. (E) PFs showing antral cavity (a) after 12 days of culturing. (F) Extrusion of oocytes (o) after 15 days of culturing. Scale bars = 50 µm.

• 

  Fig. 5 Effect of different concentrations of SNP on in vitro antrum formation in cultured buffalo PFs. The percentages were subjected to arcsine transformation and analyzed by ANOVA followed by Duncan's multiple range test. Asterisks on the bars denote significant differences compared to the control group (p < 0.05). The sample size of each group is 39, 38, 46 and 47 from left to right, respectively.

• 

  Fig. 6 Detection of apoptosis in oocytes extruded from buffalo PFs cultured in vitro. (A) Control group oocyte showing TUNEL-negative staining. (B) SNP (10-3 M)-treated PFs oocyte showing TUNEL-positive staining (nuclear brown staining; arrow). Scale bars = 50 µm.

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