Wydział Biologii

This dataset consists:

File 1 with results of the level of activin A protein in the medium from the culture of maternal knockout (m-KO), maternal-zygotic knockout (mz-KO), and wild-type mouse embryonic fibroblasts.

File 2 and 3. Two .exe file with results of in vitro maturation of activin A m-KO oocytes from Inhba^KO/LoxP Zp3-Cre females and control oocytes from Inhba^LoxP/LoxP females, including:

• File 2 with the number and percentage of mature MII stage oocytes (MII), oocytes after germinal vesicle breakdown (GVBD) and in the germinal vesicle (GV) stage, and degenerated oocytes, after 16 h of in vitro maturation;
• File 3 with the number and percentage of normal and abnormal meiotic spindles in oocytes after in vitro maturation.

File 4 with data of morphokinetics parameters from time-lapse imaging of activin A m-KO, mz-KO, and control embryos.

File 5 with the number and percentage of cell lineages cells of m-KO, mz-KO, and control blastocysts.

File 6 with results of mitochondrial activity in m-KO and control MII oocytes, based on the green to red ratio of JC-1 staining. In each experiment, the mean fluorescence intensity of oocytes was normalized to that of control oocytes, which were stained and imaged simultaneously.

File 7 with the ddPCR results of the number of Inhba transcripts in wild-type mouse GV, MII oocytes, COCs, and zygotes.

Briefly about the project and data acquisition methods:

In our project, we aimed to verify whether the lack of a phenotype in Inhba zygotic knockout embryos may be masked by the presence of maternal protein deposited in the oocyte during oogenesis. To this end, we carried out a conditional Inhba knockout in oocytes using Zp3-Cre/LoxP strategy, and received embryos with maternal knockout (m-KO), maternal and zygotic knockout (mz-KO). As a control, we used Inhba^LoxP/LoxP embryos with a floxed but functional Inhba gene.

The lack of activin A protein in Inhba mz-KO embryos was proved in the ELISA assay of the culture medium from mouse embryonic fibroblasts (MEFs) derived from Inhba mz-KO, Inhba m-KO and wild-type foetuses (File 1).

 To determine whether maternal activin A depletion affects oocyte in vitro maturation (IVM), we isolated fully grown germinal vesicle (GV) stage m-KO and control oocytes (from Inhba^LoxP/LoxP females). After 16 hours of IVM, we calculated the number and percentages of the mature MII stage oocytes (MII), immature oocytes: after germinal vesicle breakdown (GVBD) and in the GV stage, and degenerated oocytes (File 2). We then used chromatin and β-tubulin staining to assess the potential effects of maternal activin A depletion on microtubule organization, spindle poles structure, and chromosome alignment. To visualize all sides, the meiotic spindle structure was assessed based on confocal Z-stack projections (File 3).

To compare the phenotype of m-KO and mz-KO embryos with stage-matched control embryos, we analyzed the morphokinetics parameters by recording the progress of their development from the zygote to the blastocyst stage using time-lapse microscopy (File 4). The received blastocysts were immunostained to detect the presence of SOX2, SOX17, and CDX2, markers of epiblast (EPI), primitive endoderm (PE), and trophectoderm (TE), respectively. Then, using Imaris software, we counted the number and percentage of cells contributing to the cell lineages (EPI, PE, and TE) of m-KO, mz-KO, and control blastocysts (File 5).

Next, we compared the mitochondrial activity in m-KO and control oocytes based on JC-1 staining. Mitochondrial membrane potential was determined as the ratio of red to green fluorescence intensity, calculated from the sum of signal intensities across all Z-stack layers. In each experiment, the mean fluorescence intensity of oocytes was normalized to that of control oocytes, which were stained and imaged simultaneously (File 6).

Next, we used ddPCR analysis to study the Inhba mRNA levels in wild-type mouse GV, MII oocytes, cumulus-oocyte complex (COCs), and zygotes. The number of Inhba transcripts in a single ESC, oocyte, or embryo was calculated as follows: copy number/µl× dilution factor ×200 µl)/(number of ESCs or oocytes or embryos) (File 7).