Separation of Avian Preovulatory Follicle Granulosa and Theca Cell Layers for Downstream Applications

We seek to understand the specialized granulosa and theca cells of the Langhand ovary. We study differential gene expression between the two cell types at different points during ovulation. This provides insights into reproductive efficiency and fertility that will be used to sustainably enhance egg production.

Poultry Reproductive Biology is currently investigated using next generation sequencing technologies and metabolic assays. This is done to study both the reproductive tissues and the hormonal interplay involved in sexual maturity and ovulation. The Granulosa cell layer may not always come off as an intact sheet.

Patients in practice are required to ensure a higher probability of success. Extra yolk remaining on the granulosa layer is another concern. Longer incubation times in cold PBS may alleviate this issue.

Otherwise, downstream cleanup may be required. This technique will allow for standardization among avian reproductive biologists. As sequencing technology improves, elucidating the roles of specialized cells within organs becomes increasingly accessible.

Therefore, visualization of a technique that separates specialized cell types is critical for future replication. To begin, take follicles isolated from a sexually mature female bird and place them on ice. Prior to manipulation, study the follicles, noting the location of the stalk, the stigma, and the germinal disc.

Then gently roll the follicle over a dry paper towel and pull off the serosa if necessary from the follicular surface. Hold the follicle by the stalk over a bowl filled with PBS and use gravity to visualize where the stalk ends at the follicle surface. Using scissors, snip only the stalk without puncturing the follicle surface, and continue removing the serosa, until it is no longer visible.

Using the light box to illuminate, and the germinal disc as a reference, relocate the stigma on the opposite side. Gently hold the follicle with the stigma side visible and position the follicle directly over the bowl of ice cold PBS. Then, pick up the scalpel and slice gently along the length of the stigma.

Immediately drop the follicle into the PBS as the yolk begins to fall out. Using a pair of straight untoothed tissue forceps, gently peel back the pink layer of the follicular wall from the yolk on one side of the slice. Continue with small pinch and pull motions to lift the follicle wall away from the yolk.

To perform layer separation, retract approximately five millimeters of follicular wall tissue away from the yolk at a time. Once a five millimeter section has been retracted, use angled forceps in tandem with the straight forceps to probe along the follicle shell. Once the granulosa layer is located, securely, pinch it to the Theca layer and continue pulling both layers away from the yolk as previously described.

Next, use curved forceps to lightly brush the yolk away from the pinched layers. Sweep away or remove excess yolk from the bowl without scraping the granulosa layer to avoid tearing. After retracting each five to 10 millimeter section of the follicular wall, pause and use the forceps to peel the theca and granulosa layers away from each other as well as from the yolk.

Now, approach the germinal disc carefully using straight forceps. Firmly hold the granulosa layer to the Theca layer. With curved forceps, use light brushing motions to push the germinal disc away from the granulosa cells onto the yolk surface.

If the germinal disc is required as a research specimen, brush off the yolk using angled forceps. Continue separating the yolk from the follicular wall with curved forceps until the entire sphere of the follicle is devoid of yolk. Once the remainder of the yolk has been brushed away, separate the last portion of the granulosa layer from the Theca layer.

Ensure the fully separated granulosa layer is as clean as possible without being heavily coated in yoke matter. Place the granulosa and theca layers into separate containers with four degrees Celsius PBS. After placing the granulosa layer and theca layers in their designated container, gently agitate the theca layer in PBS with forceps to detach any remaining remnants of the granulosa layer.