Regulation of boar sperm functionality by the nitric oxide synthase/nitric oxide system

J Assist Reprod Genet. 2019 Aug;36(8):1721-1736. doi: 10.1007/s10815-019-01526-6. Epub 2019 Jul 19.

Abstract

Purpose: Nitric oxide (NO) is a free radical synthesized mainly by nitric oxide synthases (NOSs). NO regulates many aspects in sperm physiology in different species. However, in vitro studies investigating NOS distribution, and how NO influences sperm capacitation and fertilization (IVF) in porcine, have been lacking. Therefore, our study aimed to clarify these aspects.

Methods: Two main experiments were conducted: (i) boar spermatozoa were capacitated in the presence/absence of S-nitrosoglutathione (GSNO), a NO donor, and two NOS inhibitors, NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) and aminoguanidine hemisulfate salt (AG), and (ii) IVF was performed in the presence or not of these supplements, but neither the oocytes nor the sperm were previously incubated in the supplemented media.

Results: Our results suggest that NOS distribution could be connected to pathways which lead to capacitation. Treatments showed significant differences after 30 min of incubation, compared to time zero in almost all motility parameters (P < 0.05). When NOSs were inhibited, three protein kinase A (PKA) substrates (~ 75, ~ 55, and ~50 kDa) showed lower phosphorylation levels between treatments (P < 0.05). No differences were observed in total tyrosine phosphorylation levels evaluated by Western blotting nor in situ. The percentage of acrosome-reacted sperm and phosphatidylserine translocation was significantly lower with L-NAME. Both inhibitors reduced sperm intracellular calcium concentration and IVF parameters, but L-NAME impaired sperm ability to penetrate denuded oocytes.

Conclusions: These findings point out to the importance of both sperm and cumulus-oocyte-derived NO in the IVF outcome in porcine.

Keywords: Capacitation; In vitro fertilization; Nitric oxide; Nitric oxide synthase; Spermatozoa.

MeSH terms

  • Acrosome Reaction
  • Animals
  • Enzyme Inhibitors / pharmacology*
  • Female
  • Male
  • NG-Nitroarginine Methyl Ester / pharmacology
  • Nitric Oxide / metabolism*
  • Nitric Oxide Synthase / antagonists & inhibitors*
  • Oocytes / cytology
  • Oocytes / drug effects
  • Oocytes / physiology*
  • Sperm Capacitation / drug effects
  • Sperm Capacitation / physiology*
  • Sperm Motility / drug effects
  • Sperm Motility / physiology*
  • Swine

Substances

  • Enzyme Inhibitors
  • Nitric Oxide
  • Nitric Oxide Synthase
  • NG-Nitroarginine Methyl Ester