The Effects Of ANP On Porcine Oocyte Maturation, Fertilization And Giant Panda Spermatozoa Function

Atrial natriuretic peptide (ANP) or atrial natriuretic factor (ANF), a small peptide mainly produced in the heart, is involved in the regulation of blood pressure, salt and water excretion, cell proliferation and body fluid homeostasis. Recent reports show that ANP and its receptors are widely present in the ovary and oviduct. Generally, gondaotropins increase cAMP levels to induce oocyte maturation. Recent reports show that cGMP is involved in oocyte maturation. ANP exhibits the high affinity to NPRA thatcan activate particulate guanylate cyclase, which may play an important physiological role in reproductive tissues. In this study, we examined the effect and the mechanism of ANP on porcine oocyte maturation, fertilization and giant panda spermatozoa function.Experiment 1. This study examined the effect of atrial natriuretic peptide (ANP) on porcine cumulus-enclosed oocyte (CEO) maturation and cumulus expansion. ANP negatively regulated follicle-stimulating hormone (FSH)-stimulated germinal vesicle breakdown (GVBD; 90.1, 81.2 and 68.2% for FSH, FSH + 10 nM ANP and FSH +1 μM ANP, respectively), first polar body emission (PB1; 86.1, 75.3 and 53.3% for FSH, FSH + 1 nM ANP and FSH +1 μM ANP, respectively) and cumulus expansion (CEI; 3.47, 3.16 and 2.43 for FSH, FSH + 1 nM ANP and FSH +1 μM ANP, respectively) in a dose-dependently manner when CEOs were cultured in the maturation medium containing porcine follicular fluid (pFF). This negative effect showed a time-dependent manner after preincubation with 100 nM ANP for 5 h (78.4% PB1), 10 h (81.7% GVBD and 74.1% PB1), 20 h (78.5% GVBD and 68.9% PB1), and 44 h (75.3% GVBD and 60.5% PB1), respectively. ANP also significantly inhibited FSH-induced porcine oocyte GVBD (47.6% vs. 83.8%) and PB1 emission (22.4% vs. 45.2%) when CEOs were cultured in pFF-free maturation medium. cGMP analog 8-Br-cGMP (10 μM - 1 mM) mimicked the effects of ANP on GVBD, PB1 and CEI. The negative effect of ANP was completely reversed by KT5823 (a specific inhibitor of cGMP dependent protein kinase), while C-ANP-(4-23) (an analogue of ANP and specific binder for natriuretic peptide receptors-C) was ineffective in oocyte maturation. Both of ANP and C-ANP-(4-23) had no effect on spontaneous porcine oocyte maturation and cumulus expansion. These results suggest that ANP negatively regulates FSH-activated porcine oocyte meiotic resumption, meiotic maturation and cumulus expansion. The function of ANP on porcine oocyte maturation is via the cGMP dependent protein kinase (PKG) pathway.Experiment 2. The present study conducted experiments to examine the mechanism of ANP and forskolin on porcine oocyte maturation. The results revealed that ANP could inhibited follicle-stimulating hormone (FSH)-induced pig oocytes maturation and cumulus expansion and prevent the full phosphorylation of mitogen-activated protein kinase (MAPK) in both oocytes and cumulus cells, and that these inhibitory effects could be mimicked by 8-Br-cGMP, but blocked by a protein kinase G (PKG) inhibitor KT5823. Zaprinast, a cyclic guanosine 5'-monophosphate (cGMP)-specific phosphodiesterase inhibitor, could enhance the inhibitory effect of ANP on oocytes maturation. A specific analog of ANP, C-ANP-(4-23), which binds to the natriuretic peptide receptor C, had no effect in either FSH-induced or spontaneous oocytes maturation. Treatment with forskolin, a stimulator of adenylate cyclase, had a biphasic effect; 44 h treatment induced cumulus expansion but inhibited oocytes maturation while 2 h treatment induced maturation of cumulus-enclosed oocytes (CEOs). Both ANP and C-ANP-(4-23) could inhibit the effect of forskolin on CEOs maturation, and these inhibitory effects of ANP/C-ANP-(4-23) could be blocked by preincubation with pertussis toxin (PT), consistent with mediation by a Gi protein(s) in the cumulus cells. All these results suggest that ANP is a multifunctional regulator of FSH and forskolin on pig CEOs maturation by two signalling mechanisms respectively: one is via cGMP/PKG pathway; the other is via NPRC receptors in cumulus cells and...