Sperm-borne microRNA-34c is required for the first cleavage division in mouse

Abstract

In mammals, the sperm deliver mRNA of unknown function into the oocytes during fertilization. The role of sperm microRNAs (miRNAs) in preimplantation development is unknown. miRNA profiling identified six miRNAs expressed in the sperm and the zygotes but not in the oocytes or preimplantation embryos. Sperm contained both the precursor and the mature form of one of these miRNAs, miR-34c. The absence of an increased level of miR-34c in zygotes derived from α-amanitin-treated oocytes and in parthenogenetic oocytes supported a sperm origin of zygotic miR-34c. Injection of miR-34c inhibitor into zygotes inhibited DNA synthesis and significantly suppressed first cleavage division. A 3' UTR luciferase assay and Western blotting demonstrated that miR-34c regulates B-cell leukemia/lymphoma 2 (Bcl-2) expression in the zygotes. Coinjection of anti-Bcl-2 antibody in zygotes partially reversed but injection of Bcl-2 protein mimicked the effect of miR-34c inhibition. Oocyte activation is essential for the miR-34c action in zygotes, as demonstrated by a decrease in 3'UTR luciferase reporter activity and Bcl-2 expression after injection of precursor miR-34c into parthenogenetic oocytes. Our findings provide evidence that sperm-borne miR-34c is important for the first cell division via modulation of Bcl-2 expression.

Fig. 1.

miRNA expression in gametes and preimplantation embryos. (A) Supervised hierarchical clustering of miRNA expression. The heat map represents normalized, log-transformed relative intensities for miRNA in sperm (SP), oocyte (Oo), one-cell (1C), two-cell (2C), four-cell (4C), eight-cell (8C), morula (M), and blastocyst (B) stages. Red, green, and black colors represent high, low, and mean expression levels of miRNAs, respectively. (B) Expression pattern of cluster 1 miRNAs and miR-34c expression determined by qRT-PCR without preamplification on pooled embryos (n = 5). All values were calculated against cycle threshold (Ct) values of oocytes and are presented as relative fold-change against oocyte miRNAs [2^(Ct_oocyte − Ct_x)]. All data were normalized by endogenous RNA U6 expression. The letters “a” and “b” above bars denote P < 0.05. (C) Expression of miR-34c in oocytes, sperm, and zygotes as determined by qRT-PCR (n = 4). Five sperm, oocytes, or /zygotes were used in each experiment. (D) Expression of miR-34c in Ago2 complex of sperm. Data are presented as relative to the no-antibody control. (E) Zygotes fertilized with miR-212 precursor–transduced sperm had significantly higher levels of mature miR-212 than zygotes fertilized with scramble miRNA precursor-loaded sperm (n = 3). (F) MiR-34c precursor (Pre-miR-34c) was detected only in sperm but not in oocytes and zygotes, as determined by qRT-PCR. (G) miR-34c expression was significantly higher in zygotes and in α-amanitin–treated oocytes than in nontreated oocytes (n = 3). (H) Expression of miR-34c was significantly higher in one-cell zygotes (1C) than in oocytes (Oo) or in one-cell (1C) and two-cell (2C) parthenotes 11 h after ethanol activation. *P < 0.05; **P < 0.001.

Fig. 2.

Effects of miR-34c inhibitor on development of mouse embryos. (A) Injection of the miR-34c inhibitor suppressed zygote development (n = 5). Percentage of development is based on the number of one-cell embryos (1C) used. There were significant decreases in the development at two-cell (2C), four-cell (4C), morula (M), and blastocyst (B) stages derived from zygotes injected with the miR-34c inhibitor (▲) compared with control zygotes injected with the scramble inhibitor (■). No difference in development was found between the untreated (●) and the control zygotes. Each data point represents more than 200 embryos. The letters “a” and “b” denote P < 0.001 at the same time point. (B) The MiR-34c inhibitor inhibited pronuclei fusion. The pronuclei in zygotes injected with scramble inhibitor fused, and the zygotes cleaved 20 h after injection (magnification: 10×). (C) The percentage of embryos expressing BrdU signals was significantly lower after the injection of miR-34c inhibitor than after the injection of scramble inhibitor (n = 3). *P < 0.05 with the scramble inhibitor.

Fig. 3.

Bcl-2 is a target of miR-34c. (A) Potential miR-34c–binding region on the 3′ UTR of Bcl-2. The seed region of the miRNA is underlined. (B) The 3′UTR of Bcl-2 was cloned into reporter plasmid. The plasmid was transfected into JAR cells together with either miR-34c inhibitor or scramble inhibitor. Luciferase activity was significantly higher with the miR-34c inhibitor than with the scramble inhibitor (n = 4). *P < 0.05 with the scramble inhibitor. (C) Western blotting analysis showed that the expression Bcl-2 was elevated by inhibition of miR-34c in JAR cells. β-Actin was used as an internal control. (D) Western blotting analysis (Upper) and quantitation (Lower) of Bcl-2 protein in zygotes 6 h after injection of miR-34c inhibitor or scramble inhibitor. *P < 0.05 with the scramble inhibitor.

Fig. 4.

Effects of Bcl-2 protein on embryo development. (A) One-cell (1C) zygotes were microinjected with Bcl-2 protein (▲) or water (●). Injection of Bcl-2 protein reduced the percentage of embryos reaching the two-cell (2C), four-cell (4C), morula (M), and blastocyst (B) stages. (B) Anti–Bcl-2 antibody partly rescued the inhibitory activity of the miR-34c inhibitor (n = 3). Zygotes were microinjected with the miR-34c inhibitor (▲), Bcl-2 antibody (●), or both (■). Development percentage is based on the number of one-cell embryos used, which varied from 84–120 embryos. The letters “a,” “b,” “c,” and “d” denote P < 0.009 at the same time point. (C) Western blotting analysis indicates elevated expression of p27 in zygotes treated with the miR-34c inhibitor.

Fig. 5.

MiR-34c function is suppressed in oocytes but not in parthenogenetic oocytes. (A) MiR-34c function is suppressed in oocytes as indicated by similar levels of Bcl-2 protein expression in oocytes injected with miR-34c precursor or scramble precursor (Top) and by the similar levels of luciferase activity in oocytes coinjected with miR-34c precursor and the Bcl-2 3′ UTR luciferase reporter or control reporters (Bottom). Western blotting analysis shows no difference in Bcl-2 protein expression in normal and parthenogenetic oocytes (Middle), indicating that ethanol treatment does not affect the expression of Bcl-2 protein. (B) Western blotting analysis shows that injection of miR-34c precursor into parthenogenetic oocytes significantly suppresses Bcl-2 protein expression as compared with untreated parthenogenetic oocytes and oocytes injected with scramble miRNA precursor (Top and Middle). Coinjection of the precursor miR-34c (pre-miR-34c) and Bcl-2 3′ UTR luciferase reporter constructs but not of scramble precursor miRNA (pre-miR-Ctrl) and control 3′ UTR with mutated miR-34c–binding seed region (Ctrl 3′ UTR) suppressed Bcl-2 3′ UTR luciferase activity (Bottom). *P < 0.05; **P < 0.001.

Fig. 6.

Summary of the role of miR-34c in first cleavage. Meiosis in the testicular spermatogonia (Sp'gonia) leads to the formation of spermatocytes (Sp'cytes) and spermatids (Sp'tids), during which miR-34c is expressed in increasing amounts to enhance the expression of germ cell markers in the presence of Vasa homolog (14). The spermatids are transformed into sperm carrying miR-34c incorporating RNA-induced silencing complex (RISC). Upon fertilization, miR-34c–RISC is transferred from to the oocyte, reducing the expression of Bcl-2 and p27 and leading to S-phase entry and first cleavage. Inhibiting the process by injecting miR-34c inhibitor or recombinant Bcl-2 protein before S-phase inhibits first cleavage. Such treatment is not effective after S-phase and can be rescued partially by anti–Bcl-2 antibody.