Biphasic in vitro maturation with C-type natriuretic peptide enhances the developmental competence of juvenile-goat oocytes

Abstract

In vitro embryo production success in juvenile animals is compromised due to their intrinsic lower oocyte quality. Conventional in vitro maturation (IVM) impairs oocyte competence by inducing spontaneous meiotic resumption. A series of experiments were performed to determine if maintaining meiotic arrest during a pre-maturation culture phase (pre-IVM) prior to conventional IVM improves oocyte competence of juvenile-goat (2 months old) cumulus-oocyte complexes (COCs). In experiment 1, COCs were cultured with C-type natriuretic peptide (CNP; 0, 50, 100, 200 nM) for 6 and 8 h. Nuclear stage was assessed, revealing no differences in the incidence of germinal vesicle (GV) breakdown. In experiment 2, the same CNP concentrations were assessed plus 10 nM estradiol, the known upstream agonist activating expression of NPR2, the exclusive receptor of CNP. CNP (200 nM) plus estradiol increased the rate of oocytes at GV stage at 6 h compared to control group (74.7% vs 28.3%; P<0.05) with predominantly condensed chromatin configuration. In experiment 3, relative mRNA quantification revealed NPR2 expression was down-regulated after pre-IVM (6 h). In experiment 4, analysis of transzonal projections indicated that pre-IVM maintained cumulus-oocyte communication after oocyte recovery. For experiments 5 and 6, biphasic IVM (6 h pre-IVM with CNP and estradiol, plus 24 h IVM) and control IVM (24 h) were compared. Biphasic IVM increased intra-oocyte glutathione and decreased ROS, up-regulated DNA-methyltransferase 1 and pentraxin 3 expression and led to an increase in rate of blastocyst development compared to control group (30.2% vs 17.2%; P<0.05). In conclusion, a biphasic IVM, including a pre-IVM with CNP, maintains oocyte meiotic arrest for 6 h and enhances the embryo developmental competence of oocytes from juvenile goats.

Granulosa secreted factors improve the developmental competence of cumulus oocyte complexes from small antral follicles in sheep

“…Expansion of the extra-cellular matrix during oocyte maturation is essential for acquiring molecular machinery competence in vivo but in vitro overexpansion by promoting the hexosamine biosynthesis pathway (HBP) negatively influences in vitro oocyte competence [47,[54][55][56]. Therefore, despite higher CEI in this group, super developmental capacity was not observed.…”

“…Furthermore, markers of gap junction communication (GJA4 and GJA1) increased in the presence of CNP. Moreover, based on literature, chromatin configuration changes from non-surrounded nucleolus (NSN) to surrounded nucleolus (SN) during oocyte maturation, and this is regulated in vitro by NPs [47,48]. Recently, a novel mechanism for the CNP-induced oocyte meiotic arrest has been introduced in bovine.…”

In Vitro Production of Embryos from Prepubertal Holstein Cattle and Mediterranean Water Buffalo: Problems, Progress and Potential

“…CNP increases cGMP concentrations in COCs, which inhibits the cAMP hydrolyzing enzyme phosphodiesterase 3A, maintaining meiotic arrest [ 181 ]. Pre-maturation of prepubertal goats COCs for 6 h with CNP maintained TZP density, which is essential for cGMP transport into the oocyte and, consequently, meiotic arrest [ 182 ]. This treatment significantly increased blastocyst development rates compared to controls (30.2% vs. 17.2%), possibly due to an improved ability of the oocyte to manage oxidative stress, as CNP pre-maturation resulted in increased intra-oocyte glutathione concentrations and decreased ROS [ 182 ].…”

Effects of C-type natriuretic peptide on meiotic arrest and developmental competence of bovine oocyte derived from small and medium follicles

“…More importantly, C-type natriuretic peptide (CNP), which is expressed at high levels in mural granulosa cells, acts on the receptor natriuretic peptide receptor 2 (NPR2) in cumulus cells to generate cGMP, cyclic GMP then enters the oocyte via gap-junctional communication (GJC) and regulates the levels of cAMP by inhibiting the hydrolyzing activity of specific phosphodiesterase 3A (PDE3A), thus maintaining oocytes meiotic arrest 10 , 11 . Given the inhibitory role of CNP in the modulation of oocyte meiotic maturation, intensive efforts have focused on the development of two-step culture systems using CNP to delay or temporarily prevent spontaneous nuclear maturation, with the aim of promoting cytoplasmic maturation and subsequently improving bovine oocyte developmental competence in vitro 11 – 14 . Notably, a novel IVM system based on CNP-pretreatment, including a pre-IVM phase for 6 h using 200 nM CNP, followed by a extended IVM phase for 26–28 h, has demonstrated a remarkable improvement in bovine oocyte developmental comptence 11 .…”