Fig 1.
Experimental design.
Table 1.
Primer sequences.
Fig 2.
Developmental competency of in vitro conventional maturation of COCs derived from medium (>4 to 6 mm) compared to small (2 to ≤4 mm) size follicles.
5 replicates and minimum number of oocytes in each replicate were 50. Culture medium was conventional maturation medium containing TCM + LH (10μg/ml) + FSH (10μg/ml) + IGF (100ng/ml) + EGF (10ng/ml) + Cys (0.1mM) + FBS (15%) + E2 (1μg/ml). The asterisk represents a significant difference (P<0.05) within columns with the same color.
Fig 3.
Gene expression of AREG, EGFR, NPPC, NPR2, PGE2, and PTGS2 between cumulus cells derived from medium (>4 to 6 mm) and small (2 to ≤4 mm) size follicles at 0, 12 and 24 h post- in vitro maturation in a conventional medium.
3 replicates and minimum number of cumulus cells in each replicate were 2×105. Different superscripts demonstrate significant differences (P<0.05) within columns with the same color. The asterisks represent significant differences between the two groups at the same time of maturation. 2^-(delta CT) was presented for gene expression. AREG: amphiregulin, NPPC: natriuretic peptide precursor C, PTGS2: prostaglandin-endoperoxide synthase 2, EGFR: epidermal growth factor receptor, NPR2: natriuretic peptide receptor B, PGE2: prostaglandin E synthase 2.
Fig 4.
Determination of optimal concentration of CNP between oocytes derived from medium (>4 to 6 mm) and small (2 to ≤4 mm) size follicles.
3 replicates and the minimum number of oocytes in each replicate were 30. Different superscripts demonstrate significant differences (P<0.05). CNP: natriuretic peptide type C.
Fig 5.
Percentage of oocytes at the GV stage during 24 h IVM in four experimental groups (TCM, E2, CNP, CNP+E2).
A: medium size follicles (>4 to 6 mm) B: small size follicles (2 to ≤4 mm). 3 replicates and the minimum number of oocytes in each replicate were 20. Different superscripts demonstrate significant differences (P<0.05) within columns with the same color. TCM: tissue culture medium, E2: estradiol, CNP: natriuretic peptide type C.
Fig 6.
A, B, C: Gene expression of GJA4 and GJA1 between TCM and CNP groups in medium (>4 to 6 mm) and small (2 to ≤4 mm) size follicles. 3 replicates and the minimum number of oocytes and cumulus cells in each replicate were 30 and 2×105 cells, respectively. D: Calcein uptake through gap junctions between TCM and CNP groups in COCs derived from small size follicles (2 to ≤4 mm). 3 replicates and the minimum number of oocytes in each replicate were 10. The asterisks represent a significant difference. GJA: gap junction protein alpha, TCM: tissue culture medium, CNP: natriuretic peptide type C.
Fig 7.
Developmental competence of COCs harvested from small size follicles (2 to ≤4 mm) in sheep sequentially exposed to CNP for 6 h, then cultured in TCM+PGE2 and/or AREG for 18 h.
Different superscripts demonstrated significant differences (P<0.05) within columns with the same color. 5 replicates and the minimum number of oocytes in each replicate were 50. TCM: tissue culture medium, CNP: natriuretic peptide type C, AREG: amphiregulin, PGE2: prostaglandin E2.
Fig 8.
In vitro development of COCs derived from medium (>4 to 6 mm) compared to small (2 to ≤4 mm) size follicles that cultured in TCM (control) or TCM+NP then cultured in TCM+AREG+PGE2 (treatment).
5 replicates and the minimum number of oocytes in each replicate were 50. The asterisks represent the significant difference for blastocyst rate in small size follicles. TCM: tissue culture medium, CNP: natriuretic peptide type C, AREG: amphiregulin, PGE2: prostaglandin E2.
Fig 9.
Assessment of relative expression of pluripotency and epigenetic markers in blastocysts derived from TCM, conventional IVM (24 h), [TCM+CNP (6 h), then cultured in TCM+AREG+PGE2 (18 h)] and [TCM+CNP (6 h), then cultured in conventional IVM supplements + AREG + PGE2 (18 h)] groups.
3 replicates and the minimum number of blastocysts in each replicate were 5. 2^-(delta delta CT) was presented. The asterisks represent significant differences within columns with the same color. TCM: tissue culture medium, CNP: natriuretic peptide type C, AREG: amphiregulin, PGE2: prostaglandin E2.
Table 2.
Determination of optimal concentration of AREG in COCs derived from small size follicles.
Table 3.
Determination of optimal concentration of PGE2 in COCs derived from small size follicles.