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 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.

Methods
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 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.

Imaris data

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 100µm2 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.