Discussion Paper #1
1/ In the textbook, it is said that sperm only contributes its chromosome and centrosome to the organism during fertilization (p330). However, in the article we study, scientists found that sperm also contains miRNA, specifically miR-34c which is delivered to the oocytes during fertilization and plays an important role in zygotic division. First, they show that miR-34c (precursor and mature) comes only from the sperm, not the zygote or oocyte. The amount of pre-miR-34c was only detected in sperm by qRT-PCR method (Fig.1F). In addition, comparing the amount of miR-34c between normal zygotes and α-amanitin zygotes, or between normal zygotes and parthenotes, we could see most of the mature miR-34c in zygotes originates from sperm (Fig.1G, 1H). Secondly, they hypothesized miR-34c affects the first cell division. miR-34c inhibitor and scramble inhibitor were injected to the zygotes. Most of the zygotes with the miR-34c cannot cleave, indicating the function of miR-34c to the cell development (Fig.2). Hypothesis was further supported by the evidence that miR-34c inhibitor does not work in later cell divisions after DNA synthesis was completed. In conclusion, all the findings in the article did support the hypothesis that miR-34c is required for first cell division and changes the statement to sperm not only contribute its genetic information but also miRNA to the development of the organism.
2/
* miR-34c transferring from sperm to the oocytes functions in first cell cleavage by decreasing the expression of Bcl-2 and p27. Bcl02 is found to be a target of miR-34c since miR-34c has seeding sequence that cooperates with 3’UTR in Bcl-2. Scientists did the experiment with the Jar cells that contain transfected Bcl-2 3’UTR and either miR-34c inhibitor or scramble inhibitor. Luciferase activity and Western Blot were conducted and showed that the expression of Bcl-2 increases with the presence of miR-34c inhibitor (Fig.3). * miR-34c does not function in normal oocytes but functions in parthenogenetic oocytes. In normal oocytes, Bcl-2 level does not change when we add precursor miR-34c. However, in the parthenogenetic oocytes, it shows Bcl-2 expression decreases when injecting pre miR-34c. Injecting with scramble pre-miRNA still does not change Bcl-2 level. Therefore, miR-34c function is not suppressed in parthenogenetic oocytes (Fig.5).

3/ In figure 4, the purpose of using Bcl-2 antibody in conjunction with the miR-34c inhibitor is to show the effects of Bcl-2 protein on miR-34c inhibitor. Both proteins are related to each other. Injecting miR-34c inhibitor alone will decrease the development percentage of the embryo. Injecting Bcl-2 antibody will inhibit the Bcl-2, leading to the increase of development percentage. When we inject Bcl-2 antibody and miR-34c inhibitor together, Bcl-2 mediates the inhibitory effect of miR-34c. That is the reason why this case results to the middle line in figure 4B, indicating the average percent on embryonic development.