SMAD4 promotes somatic-germline contact during murine oocyte growth

Development of the mammalian oocyte requires physical contact with the surrounding granulosa cells of the follicle, which provide it with essential nutrients and regulatory signals. This contact is achieved through specialized filopodia, termed transzonal projections (TZPs), that extend from the granulosa cells to the oocyte surface. Transforming growth factor (TGFβ) family ligands produced by the oocyte increase the number of TZPs, but how they do so is unknown. Using an inducible Cre recombinase strategy together with expression of green fluorescent protein to verify Cre activity in individual granulosa cells, we examined the effect of depleting the canonical TGFβ mediator, SMAD4. We observed a 20-50% decrease in the total number of TZPs in SMAD4-depleted granulosa cell-oocyte complexes, and a 50% decrease in the number of newly generated TZPs when the granulosa cells were reaggregated with granulosa cell-free wild-type oocytes. Three-dimensional image analysis revealed that TZPs of S..., Counting the number of TZPs that project to an oocyte
To quantify the number of F-actin-stained-TZPs, a confocal optical section was obtained at the equatorial plane of the oocyte. Using Fiji software (National Institutes of Health, Bethesda, MD), a segmented circle was drawn around the oocyte in the middle of the zona pellucida, and the fluorescence intensity at each point on the line was obtained. Each point whose value was above the background value of the oocyte cytoplasm and higher than each of its immediately neighboring points was counted as a TZP.
After the fluorescence intensities for each point were obtained and entered into an Excel spread sheet, the following formula was used in Excel to count the number of TZPs:
=COUNTIFS(B5, \">\"&$C$14,B5, \">\" &B4,B5, \">\" &B6)
This formula is entered in each cell of a column (for example, column D). B5 is the intensity value at position B5 in the spreadsheet. It will be counted as a TZP if its value is greater than the ..., , # SMAD4 promotes somatic-germline contact during murine oocyte growth

[https://doi.org/10.5061/dryad.tht76hf6s](https://doi.org/10.5061/dryad.tht76hf6s)

Data reports characteristics of transzonal projections in intact granulosa-oocyte complexes, denuded oocytes from which the granulosa cells were removed immediately before fixation, and reconstructed granulosa cell-oocyte complexes.

## Description of the data and file structure

Data are provided in spreadsheets in csv format.

**The file labeled 'TZPs denuded' is organized as follows:**

Columns A-D compare data of controls (A, B), where the cells carried floxed alleles of Smad4 but did not carry the Cre transgene, to presumptive knockouts (KO), where the cells carried floxed alleles of Smad4 and the Cre transgene (C, D).

Columns A and C show the diameter (um) of the oocyte. Columns B and D show the number of TZPs associated with that oocyte, corrected for the diameter of the oocyte.

Column E is a spacer column, inserted for clari...