Table 1.
Primer detailed information for each gene analyzed.
Fig 1.
Effect of pre-IVM with CNP and estradiol on the maintenance of meiotic arrest in juvenile-goat oocytes.
(A) Germinal vesicle (GV) rate of oocytes cultured for 6 h and 8 h with different CNP concentrations. A total of 47–48 oocytes were stained per condition (4 replicates). (B) GV rate of oocytes cultured for 6 h and 8 h with different CNP concentrations plus estradiol. A total of 46–50 oocytes were assessed per condition (4 replicates). Each bar represents mean + SEM. Different superscript letters (a-c) in each column indicate statistically significant differences (P<0.05). (C) Relative gene expression of natriuretic peptide receptor (NPR2) in COCs after 6 h of pre-IVM with CNP and E2. Five samples were tested per group (10 COCs per sample). Each bar represents relative quantification (RQ), and error bars show RQ max and RQ min. Superscript symbols indicate statistical differences relative to control 0 h: (*) P<0.001; (**) P<0.0001.
Fig 2.
Effect of pre-IVM with CNP and estradiol for 6 h on germinal vesicle chromatin configuration of juvenile-goat oocytes.
Germinal vesicles (GV) are classified as GV1: diffuse filamentous chromatin; GV net-like: condensed net-like chromatin; GV clumped: condensed clumped chromatin; GVBD: broken nuclear membrane. A total of 46–50 oocytes per treatment were assessed (4 replicates). Each bar represents mean + SEM. Different superscript letters (a-c) in each column indicate statistically significant differences (P<0.05).
Fig 3.
Effect of pre-IVM with CNP and estradiol on the transzonal projections (TZPs) density of juvenile-goat COCs.
(A) Phalloidin-FITC Average fluorescence intensity in the zona area of COCs after recovery (control 0 h), 6 h of IVM, 6 h of pre-IVM with CNP and E2, 24 h of IVM, and 6 h of pre-IVM followed by 24 h IVM. At least 32 COCs were assessed per group (4 replicates). Each bar represents mean + SEM. Different superscript letters (a-c) in each column indicate statistically significant differences (P<0.05). (B) Representative confocal images. Positive actin filaments are observed as continuous filaments going from the cumulus cells to the oocyte through the zona region.
Fig 4.
Nuclear maturation rate of juvenile-goat oocytes after biphasic IVM (6 h pre-IVM with CNP and estradiol, followed by 24 h IVM) and control IVM (24 h).
Nuclear stage was classified as GV: germinal vesicle; MI: metaphase I; MII: metaphase II. A total of 40 oocytes were assessed per group (4 replicates). Each bar represents mean + SEM.
Fig 5.
Mitochondrial DNA copy number in juvenile-goat oocytes after recovery from the follicle (control 0 h), biphasic IVM (6 h pre-IVM with CNP plus estradiol, followed by 24 h IVM) and control IVM (24 h).
A total of 30 oocytes per group were assessed (3 replicates). Each bar represents mean + SEM.
Fig 6.
Effect of biphasic IVM on GSH and ROS levels of juvenile-goat oocytes.
(A) H2DCF-DA-ROS average fluorescence intensity per oocyte after biphasic IVM (6 h pre-IVM with CNP and E2, followed by 24 h IVM) and control IVM (24 h) and representative images. (B) MCB-GSH average fluorescence intensity per oocyte following biphasic and control IVM and representative images. A total of 30 oocytes were assessed per group (3 replicates). Each bar represents mean + SEM. Different superscript letters (a, b) in each column indicate statistically significant differences (P<0.01).
Fig 7.
Relative gene expression of BMP15, DNMT1, GDF9, FSHR, PTX3 and TNFAIP6 in juvenile-goat COCs after biphasic IVM (6 h pre-IVM with CNP and E2, followed by 24 h IVM), control IVM (24 h), and oocyte recovery (control 0 h).
Five samples were tested per group (10 COCs per sample). Each bar represents relative quantification (RQ), and error bars show RQ max and RQ min. Superscript symbols indicate statistical differences relative to control 0 h: (*) P<0.05; (**) P<0.001; (***) P<0.0001. Different superscript letters (a—b) indicate statistical differences between treatment groups (P<0.05).
Fig 8.
Blastocyst development and quality of juvenile-goat oocytes after biphasic IVM (6 h pre-IVM with CNP plus E2, followed by 24 h IVM) and control IVM (24 h).
(A) Cleavage rate (48 hpf) and blastocyst rate (8 dpf) per number of oocytes (N). A total of 196–198 oocytes were cultured per group (5 replicates). (B) Cell count of the inner cell mass and the trophectoderm of expanded and hatched blastocysts. A total of 22–33 blastocysts were analyzed per group (4 replicates). Each bar represents mean + SEM. Different superscript letters (a-b) in each column indicate statistically significant differences (P<0.01).