Molecular regulation of miR-378 on the development of mouse follicle and the maturation of oocyte in vivo

Figures & data

Table 1. Primers used for quantitative-PCR amplification.

Genes	Sequences of primers	Production(bp)	Genbank
GAPDH	F:5＇- GTGTTCCTACCCCCAATGTG −3＇	299	XM_017321385
	R:5＇- GTCATTGAGAGCAATGCCAG −3＇
Bax	F:5＇- AGACAGGGGCCTTTTTGCTAC −3＇	137	NM_007527
	R:5＇- AATTCGCCGGAGACACTCG −3＇
Bcl-2	F:5＇- GCTACCGTCGTGACTTCGC −3＇	147	NM_177410
	R:5＇- CCCCACCGAACTCAAAGAAGG −3＇
BMP-15	F:5＇- TGGGGAGTGGTGCTTTTTATG −3＇	93	NM_009757
	R:5＇- GGGCAATGTAGGGTCGTCAG −3＇
GDF-9	F:5＇- GTCACCTCTACAATACCGTCCG −3＇	119	NM_008110
	R:5＇- CACCCGGTCCAGGTTAAACA −3＇
c-Kit	F:5＇- GCCTGACGTGCATTGATCC −3＇	110	NM_001122733
	R:5＇- AGTGGCCTCGGCTTTTTCC −3＇
Cx-43	F:5＇- ACAAGGTCCAAGCCTACTCCA −3＇	145	NM_010288
	R:5＇- CCGGGTTGTTGAGTGTTACAG −3＇
Cx-37	F:5＇- TCCCACATCCGATACTGGGT −3＇	91	NM_008120
	R:5＇- CCCGCCGAGACAGGTAGAT −3＇

Figure 1. MicroRNA-378 disrupted the mouse estrous cycle. (a) The expression of miR-378-3p and miR-378-5p determined by quantitative PCR. (b) Estrous cycle after ovary-subcutaneous injection. The mice were injected at dioestrum. They were at the same stage of estrous cycle over the next 2–3 d, then the estrus of mice with miR-378 overexpression lasted much longer than that of control group (4 d in miR-378 group and 2 d in GFP group). (c) Aromatase expression after microinjection of GFP control, miR-378, Inhibitors and miR-378+ inhibitors determined by western blotting. Data are Mean ± SD of at least 3 independent experiments; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2. Effects of miR-378 on ovary development. (a) Ovarian size and morphology. (b) Index of ovary/body weight. (c) The mRNA ratio of Bax/Bcl-2 determined by quantitative PCR. (d) Bax and Bcl-2 protein expression determined by western blotting. Data are Mean ± SD of at least 3 independent experiments; *P < 0.05; **P < 0.01.

Figure 3. Influence of miR-378 on follicle development. (a) Morphological and histochemical analysis by HE staining. (b-d) Percentages of primordial follicles, secondary follicles and antral follicles with different diameter were counted after HE staining. (e, f) The mRNA expression of BMP-15 and GDF-9 determined by quantitative PCR. Data are Mean ± SD of at least 3 independent experiments; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4. MicroRNA-378 decreased the oocyte-granulosa interaction. (a-c) The mRNA expression of c-Kit, Cx-43 and Cx-37 determined by quantitative PCR. (d, e) The protein expression of Cx-43 and Cx-37 determined by western blotting. Data are Mean ± SD of at least 3 independent experiments; *P < 0.05; **P < 0.01.

Figure 5. The immunofluorescence of Cx-43. (a)Histological sections are stained red with anti-Cx-43 antibody, nuclei are stained blue with Hoechst33342. (b) Percentage of Cx43 positive cells. Data are Mean ± SD of at least 3 independent experiments; *P < 0.05.

Figure 6. MicroRNA-378 reduced the percentage of oocyte maturation. (a) Oocytes after IVM 16–18 h. (b) Percentage of IVM after different treatments. (c) Normal and abnormal spindle assembling of oocytes. (D) The ratio of abnormal spindle assembling of oocytes after different treatments. (e) The treated female mice were maintained at normal condition and were mated with normal male mice. After birth, the average number of pups was counted. (f) The compared number of pups/litter in each batch of the paired breeding. Data are Mean ± SD of at least three independent experiments; *P < 0.05; **P < 0.01; ***P < 0.001.