DOI: 10.5061/dryad.tht76hf6s
title: Data from: SMAD4 promotes somatic-germline contact during murine oocyte growth

description: 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
 SMAD4-depleted cells were also longer than controls and more frequently
 oriented towards the oocyte. Strikingly, the transmembrane proteins,
 N-cadherin and Notch2, were reduced by 50% in these cells. SMAD4 may thus
 modulate a network of cell adhesion proteins that stabilize the attachment
 of TZPs to the oocyte, thereby amplifying signalling between the two cell
 types.
description: <strong>Counting the number of TZPs that project to an
 oocyte</strong> 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,
 "&gt;"&$C$14,B5, "&gt;"
 &B4,B5, "&gt;" &B6) This
 formula is entered in each cell of a column (for example, column D). B5 is
 the intensity value at position B5 in the spreadsheet. It will be counted
 as a TZP if its value is greater than the background ($C$14 in this sample
 formula), and greater than the intensity at the preceding position (B4),
 and greater than the intensity at the following position (B6). The value
 at B6 will be compared to those at B5 and B7, and so on. The value at C14
 remains constant, since the same background value is applied in all
 cases. Excel will assign a value of 0 or 1 to each cell
 in column D, based on the result of the calculation. The sum of the values
 in column D is the number of TZPs. <strong>Imaris
 data</strong> To individually segment granulosa
 cells and their corresponding TZPs, Imaris 9.7.2 Software (Bitplane) was
 used. First, z-stacks of 0.2 μm thickness covering the middle region of a
 follicle were imaged to obtain a conventional 2D Maximum Intensity
 Projection (MIP). Then, a 3D rendered segmentation of GFP-positive
 granulosa cells was created with the “Cell detection” tool   where cell
 boundaries were detected based on GFP membrane staining. Smallest diameter
 was set to 100 µm, membrane detail of 0.2 μm, and the threshold was
 adjusted based on local contrast. Segmented cells with a volume of less
 than 100µm<sup>2</sup> were deleted and fusing or fragmented
 cells were edited using the “merge” or “split” tools to match the original
 image. The oocyte was also segmented using the phalloidin red channel.
 After edition, cells were converted to surface objects with the tool
 “Convert to surfaces” to record their measurements. To render individual
 TZPs, the “Filament tracer” tool and semi-automated tracing with the tool
 “Autopath” was used to draw the path of each TZP from the granulosa cell
 body (start) to the oocyte surface (end). TZP diameter (thickness) was
 calculated automatically with a minimum diameter of 0.1 μm. To determine
 the distance of the TZP tip to the oocyte surface, the segmented TZP was
 automatically divided into discrete spots using the extension “Filament to
 Spots”. This allowed the calculation of the distance from each spot to the
 oocyte surface. All measurements were exported using the “distance to
 oocyte surface” from each TZP spot. To determine the TZP orientation (away
 or towards the oocyte), the distance of the spot located at the TZP start
 was subtracted from the distance of the spot located at the TZP tip. A
 positive difference indicates that the TZP tip is closer to the oocyte and
 a negative difference indicates that the TZP tip is farther from the
 oocyte.
description: # 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 clarity. Columns F-I compare data of controls (F, G), where the cells carried wild-type (wt) alleles of Smad4 and the Cre transgene, to presumptive knockouts (KO), where the cells carried floxed alleles of Smad4 and the Cre transgene (H, I). Independent biological replicates are identified. For each replicate, the number of TZPs in both control and KO groups was normalized to the mean number in the control group. This allowed results from different replicates to be compared. Blank lines separate the replicates for clarity. Note that there are not necessarily the same number of specimens in the control and KO groups in each replicate. Null has been entered in blank cells to avoid confusion. **The file labeled 'TZPs GOCs' is organized in the same way, except as follows.** The controls in rows 1-27 are cells that carried wild-type (wt) alleles of Smad4 and the Cre transgene. The controls in lines 29-63 carried floxed alleles of Smad4 but did not carry the Cre transgene. They have been put in the same column because the data were plotted together in Fig. 2B of the manuscript. **Data obtained from analysis using Imaris of intact GOCs (Imaris intact GOC) and reaggregated GOCs (Imaris reaggregated GOC) are presented as follows.** The title of each file indicates the parameter that was quantified using Imaris. For each parameter, data from controls that carried wild-type alleles of Smad4 and the Cre transgene is shown in the left column (indicated by 'wt' in the label in the top row), and data from presumptive knockouts, where the cells carried floxed alleles of Smad4 and the Cre transgene, is shown in the right column (indicated by 'fl/fl' in the label in the top row). GC refers to granulosa cell. TZP refers to transzonal projection. The following parameters were recorded. Volume of GCs. Each value corresponds to an individual cell. Sphericity of GCs. Each value corresponds to an individual cell. Volume of GCs selected for analysis. Each value corresponds to an individual cell. Relative number of TZPs per granulosa cell, corrected for cell volume. TZP length. Each value represents an individual TZP. TZP width. Each value represents an individual TZP. *The following parameters were recorded only for the reaggregated GOCs.* Distance to oocyte of tip of TZP relative to origin of TZP Proportion of TZPs that are not connected to an oocyte. ## Sharing/Access information Not applicable. ## Code/Software Imaris was used for analysis of some of the data, as described in the publication. Data are presented as csv files.