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

Summary

Here, we describe a protocol for separating yolk, granulosa cells, and theca cells in avian preovulatory follicles. This precision handling enables critical investigations into the role of these layers in reproductive function, aiding the understanding of follicular development, hormonal regulation, and disease research for enhanced agricultural yield and biomedical insights.

Abstract

Layer hens (egg-laying chickens) and broiler breeders (breeding stock for meat-producing chickens) are crucial to the world's food supply as a reliable source of protein. They are also an emerging animal model for the study of human reproductive disease. As the field of poultry research develops, the health and function of the layer hen and broiler breeder ovary will be an important point of study for both agricultural and biomedical researchers. One of the challenges presented by this emerging interest is the need for replicable techniques that all researchers can employ in ovarian specimen collection. In particular, a detailed visual process must be established to define the proper separation of the specialized granulosa and theca cell layers from hen follicles to achieve agreement and consistency among researchers.

This study describes the extraction of preovulatory follicles and ovary tissue in white leghorn hens of prime reproductive age. The separation of these follicles is performed under cold, liquid conditions to congeal the yolk for easier manipulation and to prevent the follicle's own weight from tearing apart cell layers during the separation process. Once the separation is complete, the desired cell layers can be further digested for tissue culture approaches or can be cryopreserved for genomic and proteomic analyses.

Introduction

Egg-producing chickens are a key component of the world food supply chain and are an evolving animal model for the study of fertility and ovarian cancer. The chicken has a long history of laboratory use, from in ovo studies of influenza vaccines¹ to the study of cancerous tumor growth², and is a key animal for insight into gonadal development^3,4,5. Understanding the role of a hen's reproductive system, in particular, the cyclical nature of ovarian follicular development and the types of cells involved, is an area of great interest and potential application. Multi-omics approaches to interrogate the hen ovary have applications for increasing agricultural yield through productivity analysis and elucidating factors toward human health by way of the hen as a model for reproductive disease.

The avian ovary has very distinct developmental follicular stages composed of (1) small primordial and primary follicles (>1 mm), (2) variably sized pre-recruitment follicles (white to pale yellow, 2-8 mm), and (3) large, yolk-filled preovulatory follicles (yellow) (Figure 1)^5,6,7. A sexually mature avian ovary is composed of an outer cortex that houses the ovarian follicles and the inner medulla composed of smooth muscle, nerves, and vasculature⁸. The entire cycle of follicular development takes place in the cortex. It is not until approximately five months of age, however, that only the left ovary demonstrates a full developmental hierarchy of preovulatory (i.e., yellow) follicles, and the chicken will undergo her first ovulation and oviposition (egg lay). The preovulatory follicles establish a grossly visible hierarchy of approximately 5 to 6 follicles that range from the largest and next to ovulate - F1 follicle - to the lesser developed, smallest follicle- F5 or F6. Preovulatory follicles are easily distinguishable from the smaller pre-recruitment follicles due to their larger size, vast innervation, and deep-yellow yolk.

All follicles contain specialized cell types that support varied roles in signaling, growth, and development of the follicle as it progresses through different stages prior to ovulation - these are the granulosa cells, theca cells, and oocytes (female gametes) (Figure 2A). In particular, the granulosa cell layers create the inner wall of the follicle, and the theca cells form the other wall surrounding the oocyte. As a follicle progresses through the preovulatory hierarchy, the theca and granulosa cell layers begin to thin out, particularly along a point directly across from the stalk of the preovulatory follicle. The stalk is the follicle's point of attachment to the ovary (Figure 2B). This line of the granulosa and theca layers opposite the stalk is called the stigma (Figure 2C). It entirely lacks vasculature and will be the point of follicular rupture during ovulation of an F1 follicle. Also visible is the germinal disc, or the fluffy white layer of support cells that surround the oocyte (Figure 2B).

Both granulosa and theca cells are key components of follicle development as they surround and support the oocyte through complex hormonal signaling. The granulosa cell layer supports steroidogenic pathways via cyclic AMP (cAMP) signaling to bring about progesterone production^9,10,11. Theca cells, on the other hand, temporally produce estradiol, which is largely different than in mammals whose theca cells produce androgen and progesterone^12,13,14. Taken together with external signaling, the granulosa and theca cells are crucial drivers in follicle maturation and oviposition. The technique of separating the granulosa and theca layers was originally reported by Gilbert et al. in 1977¹⁵. Achieving a comprehensive separation of the granulosa and theca layers and ensuring no granulosa material remains behind can present difficulties with the Gilbert method. Furthermore, the absence of a clear visual guide for completing the separation can render the task challenging to conduct. This study aims to describe the extraction and separation of granulosa, theca, and oocyte layers from the preovulatory follicles, offering adaptations and delivering a clear visual representation through the use of both images and videos (Figure 3). Visualizing this technique will allow scientists studying avian ovarian follicles to reproducibly capture granulosa and theca layers for both agricultural and biomedical multi-omics approaches.

Protocol

All animals used in this study were maintained and euthanized per the protocol approved by the University of Delaware's Institutional Animal Care and Use Committee (IACUC Protocol 110R).

1. Preparation

1. Sterilization
   1. Autoclave 5 oz glass bowls using gravity displacement autoclaving (121 °C for 20 min) covered with aluminum foil with autoclave tape atop to maintain and indicate sterility.
      NOTE: One bowl will be needed for each follicle the separation is being performed on.
   2. Autoclave dissection tools using gravity displacement autoclaving (121 °C for 20 min), including scissors, curved forceps, and straight forceps in sterilization pouches.
2. Setup of separation area
   1. Set up follicle separation area as seen in Figure 4. Fill holding containers with ice just prior to follicle separation and replenish as necessary throughout the follicle separation process.
3. Solutions
   1. Dilute to 10x PBS concentration to 1x working stock and place in +4 °C refrigerator the day prior to dissection.
   2. Dilute isopropanol to 70% working stock concentration in the spray bottle.

2. Dissection

1. Euthanize a sexually mature female bird.
   1. Choose a sexually mature female bird (must be currently laying eggs) and euthanize¹⁶ by approved method (AVMA Guidelines) as per institutional care and use protocol, such as cervical dislocation.
   2. For the capture of the largest F1 follicle, euthanize the animal within the first 4 h of light onset for the day. Palpate the lower abdomen prior to euthanasia to confirm a hard-shelled egg in the uterus; this ensures that oviposition and, thus, the next ovulation has not yet occurred.
2. Access the ovary.
   1. Spray the external carcass and feathers with 70% isopropanol. This dampens the feathers and cleans the incision point.
   2. Using dissection scissors and a gloved hand, pinch and cut a single horizontal (transverse) midline incision approximately 7.5 cm (3 inches) long. Pinch the skin, muscle, and fat layers up prior to incision to avoid puncture of organs.
   3. Widen the incision laterally on the bird's right side and probe with gloved fingers to push visceral organs out of the way of the scissors. Continue until 2-3 of the animal's ribs have been cut on the right side.
   4. Widen the incision toward the left side of the bird as done on the right. Proceed with added caution as the preovulatory follicles of the ovary are located here, protrude from the ovary surface, and can easily rupture. Use gentle pressure with fingers to keep preovulatory follicles away from the scissors as 2-3 ribs are cut on the bird's left side.
   5. Place one hand on the lower abdomen of the bird and the other firmly on the underside of the breastbone, and manually retract the breast of the bird upward to allow further access to the coelomic cavity of the bird.
   6. Holding each of the bird's thighs close to the hock joint, manually push them backward to dislocate both hip joints simultaneously. There is now complete access to the ovary both visually and spatially.
3. Remove preovulatory follicles.
   1. Perform all steps by routine visual sight. If an individual has trouble visualizing at any stage, use a dissecting microscope.
   2. Using only gloved fingers, gently cup each visible yellow preovulatory follicle and extract it from the ovary. Only pinch at the stalk during the extraction to assist in severing the stalk. Do not pinch the follicle itself, or it may rupture.
   3. Place each follicle into the sterile container with ice-cold PBS.
   4. Leave preovulatory follicles on ice for at least 5 min to allow for congealing of the yolk. The warm yolk will adhere to the surrounding follicular cell layers, making the subsequent separation techniques difficult to perform.

3. In-lab separation

1. Take an isolated follicle from the sterile container and place the container with the remaining follicles back on ice. Study the follicle prior to manipulation, taking note of the location of three features: the stalk, the stigma, and the germinal disc (Figure 2).
2. Begin by removing the serosa from the follicular surface. Roll the follicle gently over a dry paper towel to remove the serosal layer, gently pulling it off if necessary.
3. Hold the follicle by the stalk over the bowl filled with PBS. Use gravity to visualize where the stalk ends at the surface of the follicle and, using scissors, snip only the stalk without puncturing the follicle surface. Continue with the removal of the serosa until it is no longer visible.
4. Using the lightbox to illuminate and the germinal disc as a reference, relocate the stigma on the opposite side and gently hold the follicle with the stigma side visible. Hold the follicle directly over the bowl of ice-cold PBS.
5. Pick up the scalpel, slice gently along the length of the stigma, and immediately drop the follicle into the PBS as the yolk begins to fall out. Do not hold the follicle tightly, as this will push the yolk out instead of allowing it to simply fall out through gravity.
6. 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. Complete this extraction using little pinch-and-pull motions that lift the follicle wall away from the yolk.
7. Layer separation works best when approximately 5 mm of follicular wall tissue has been retracted away from the yolk at a time. When a 5 mm section of the follicular wall is retracted successfully from the yolk, use angled forceps in tandem with the straight forceps to probe along the follicle shell.
8. In some instances, the granulosa layer may be more closely associated with the yolk than the theca layer. In this situation, simply probe very gently across the surface of the yolk using either pair of forceps to try to pull the tissue-paper-like layer away from the yolk. Once the granulosa layer has been located, pinch it securely to the theca layer and proceed with pulling both layers away from the yolk as previously described.
9. Use the curved forceps to lightly brush the yolk away from the pinched layers. Sweep away or remove excess yolk from the bowl. Do not scrape the granulosa layer; this will cause tearing. Vigorous motions will cloud the PBS solution and reduce visualization. Proceed with careful and deliberate motions.
10. After each 5-10 mm section of follicular wall is retracted, pause to take the theca and granulosa layers in each set of forceps and ensure that the two layers are being peeled away from each other gradually, just as they are both being peeled away from the yolk. Continue this process around the outside of the follicle. If manipulation becomes difficult due to the flap of the granulosa-theca layer, turn the follicle to begin the process from the other side of the slice.
11. During the process of separation, the germinal disc (female gamete and germinal vesicle) will be encountered. This is an area that is extremely adherent to both the granulosa layer and the yolk. Approach the germinal disc carefully, using the straight forceps to firmly hold the granulosa layer to the theca layer, as the curved forceps are used in light brushing motions to push the germinal disc away from the granulosa cells onto the yolk surface.
    1. If the germinal disc is desired as a research specimen, brush off the yolk at this point with angled forceps and store as desired.
12. Continue separating the yolk from the follicular wall with the curved forceps until the entire sphere of the follicle has had its yolk removed.
13. Once the remainder of the yolk has been brushed from the layers, separate the last portion of the granulosa layer from the theca layer. The fully separated granulosa layer may not be entirely devoid of yolk matter, but ensure it is not coated with it. Place the granulosa layer and thecal layer into separate containers with 4 °C temperature PBS.
    1. Once the granulosa layer is in a separate container from the theca layer, gently agitate the theca layer back and forth in the PBS with forceps to ensure any remaining remnants of the granulosa layer detach.
       NOTE: Certain processing methods post-separation may account for the removal of lipid debris by digestion or centrifugation, depending on the desired study.
    2. When first practicing this technique, prepare and analyze representative separated specimens histologically to confirm accurate layer separation and collection.
       1. To do this, fix portions of separated tissues in neutral buffered formalin using mesh tissue cassettes or sponges to prevent specimen loss, then paraffin-embed, section, and stain the section with Hematoxylin and Eosin (H&E).
       2. Due to its delicate nature, carefully handle and place the granulosa layer onto biopsy sponges in a histological cassette to ensure the cellular layer stays intact.
       3. Put the theca layer directly within a histological cassette. Doing so will also allow visualization of the level of specimen contamination by other follicular tissue (i.e., granulosa cell layer specimen with adherence of yolk protein or thecal cells).
          NOTE: It may not always be possible to obtain only a single layer of interest due to the close association of follicular tissues. However, knowing the level of possible contamination can inform the interpretation of subsequent multi-omics approaches.

Results

This protocol yields the separation of granulosa and thecal cell layers from the ovarian follicle (Figure 5 and Figure 6). These layers can then be prepared for histological examination to confirm successful separation. These histological images, reviewed and captured by a board-certified veterinary pathologist (EMB), clearly demonstrate the effective separation of the granulosa and theca layers for both the F1 and F5 preovulatory follicles (Figure 7). Notably, the granulosa layer exhibits intricate folds, a consequence of its fragile nature after extraction and separation from adjacent thecal tissue. Granulosa cells are identifiable by their columnar to pseudostratified columnar shape, featuring close attachment to one another and distinct cell membrane borders (Figure 7A,B).

The theca cell layer is characterized by spindle-shaped cells with small, dense nuclei and may include large luteal cells (enlarged polygonal cells with abundant clear to foamy cytoplasm indicative of active steroid hormone production). Blood cells (nucleated ovoid cells in avian species) and small caliber vessels are common in the thecal layer and are to be expected. The underlying ovarian stroma will also likely be visible with the thecal layer sample (Figure 7C,D)⁸. Stromal tissue tends to show decreased cellular density compared to the thecal layer and can be visualized by an abrupt change in cellular orientation from circumferential (cells align in layers parallel to one another and circumferential to the follicular lumen) to multidirectional (cells align in many directions) In cases where the granulosa cells are retained with the theca layer, a thin layer of columnar granulosa cells will be evident immediately above the theca cells (Figure 8). To ensure complete layer separation, it is crucial to thoroughly observe the entire inverted theca layer. Step 3.10.1 identifies an additional rinse step to also ensure that there are few to no granulosa cells remaining after layer separation. In conclusion, the histological examination confirms the successful separation of the granulosa and thecal layer from the preovulatory follicle, highlighting the distinct characteristics of each layer and validating the proposed separation method.

Figure 1: Ovary, mature laying hen. Hen ovary with all follicles attached. Preovulatory follicles are labeled (F1-F5) to demonstrate an approximate size comparison from the largest (F1) to smallest (F5 or F6) follicles. Some hen ovaries may have up to six preovulatory follicles (not shown). Please click here to view a larger version of this figure.

Figure 2: Ovarian preovulatory follicle anatomy, mature laying hen. (A) A schematic of the cellular layers with cell types and important tissue components that comprise the preovulatory follicle (created using BioRender). Note that the figure is not to scale. (B) Gross identification of the stalk (point of follicular attachment to the ovary) and germinal disc (site of female gamete) on the follicular structure. (C) Gross identification of the stigma on the follicular structure. Please click here to view a larger version of this figure.

Figure 3: General workflow of ovarian follicular collection and separation in poultry. A schematic of the process to obtain and collect cellular layers (created using BioRender). The process begins with obtaining sexually mature hens and extracting their ovarian follicles. These follicles are then collected in a container with 1x PBS and kept on ice. A single follicle is then transferred to a dish containing 1x PBS on ice, where a lightbox is utilized for separating the cellular layers. Once separation is achieved, researchers can utilize the sample for various purposes, including DNA analysis, RNA analysis, protein analysis, cell culture, or histology. Please click here to view a larger version of this figure.

Figure 4: In-laboratory setup for ovarian follicular separation. A lightbox and dissecting microscope are required for proper visualization. For optimal preservation of samples, glass bowls and PBS should be pre-chilled and kept on ice until separation is complete. A sterile glass dish, collection container, scalpel blade, and dissection tools will be needed. It is recommended also to have a sheet to record how well the separation occurs. Please click here to view a larger version of this figure.

Figure 5: Initial separation of follicular layers in Layer hen ovaries. Forceps are used to hold onto both the theca (pink upper layer) and the granulosa (thin-white lower layer) layers. At this point, in the separation process, both layers of the follicular wall are still partly adhered to each other. Please click here to view a larger version of this figure.

Figure 6: Representative gross anatomic separation of ovarian follicular layers in poultry. (A) Isolated granulosa layer. (B) Isolated theca layer. Please click here to view a larger version of this figure.

Figure 7: Representative histologic separation of F1 & F5 granulosa and theca layers from ovarian tissue in Layer hens. Histologic identification of isolated granulosa layer from the (A) F1 and (B) F5 preovulatory follicles at 400x magnification. Isolated theca cellular layer from the (C) F1 and (D) F5 preovulatory follicles at 400X magnification. Granulosa cells exhibit a columnar to pseudostratified columnar shape with distinct cell membrane borders, while theca cells display spindle-shaped morphology with small, dense nuclei. Additionally, the presence of blood cells and small caliber vessels, along with the underlying ovarian stroma, is commonly observed in the thecal layer sample. Please click here to view a larger version of this figure.

Figure 8: Retention of granulosa layer following separation of follicular layers from poultry ovaries. In a small number of isolations, patchy retention of granulosa cells (top) can be observed partially attached to the thecal layer (middle), overlaying the follicular stroma (bottom), highlighting the need for precise handling and separation for pure cell layers. The image is at 200x magnification. Please click here to view a larger version of this figure.

Discussion

This study outlines the procedure for the distinct separation of both granulosa and thecal cell layers from preovulatory follicles in poultry. Unlike the existing method established by Gilbert et al. in 1977, the adaptations made in this method allow for a more controlled environment when manually separating the granulosa cell layer from the theca layer from all preovulatory follicles¹⁵. Furthermore, it enables researchers to visualize the granulosa cell layer throughout the whole procedure, enhancing precision and analytic capabilities. This technique can readily be adapted for isolating granulosa or theca layers for other poultry species and could potentially be used for other oviparous (egg-laying) animals^17,18. This procedure provides researchers with the opportunity to isolate and examine the distinct follicular cell layers, interrogation of which will allow for valuable insights into research areas as diverse as agricultural poultry production, avian reproduction, and potential modeling of human fertility and reproductive disease¹⁹.

When performing this protocol, there are several critical steps to consider. The first is allowing the follicle to cool to approximately 4 °C to help congeal the yolk before cutting along the stigma. This is performed by immediately immersing the follicles in ice-cold PBS and maintaining these cold temperatures after removal from the chicken. The longer the follicle is cooled in ice-cold PBS, the easier the layer separation is. Different downstream multi-omics applications may require quicker handling to prevent degradation of molecules of interest; however, it is strongly recommended that the follicle be on ice for a minimum of 5 min^20,21. Additionally, it is important to take precautions while removing each preovulatory yellow follicle from the ovary during dissection because puncture of a single preovulatory follicle will not only lead to incorrect identification of the follicle hierarchy but will also make it harder to achieve complete separation of cell layers for that follicle.

Likewise, prior to any follicular incision, vasculature must be removed from the outside of the follicle as gently as possible to avoid puncture of the follicle⁸. During follicular wall layer separation, the initial identification of the granulosa cell layer may be difficult as it will either be adherent to the thecal layer or the yolk. The most effective method for initial visualization of the adherent granulosa layer is to first observe the cut edges of the theca layer on either side of the follicular incision. Probe gently along the surface with closed forceps to gently peel back the adherent layer. If the granulosa cell layer is still unidentifiable, inspect the section of yolk that would have been adjacent to the follicular incision line and gently probe the yolk surface in short, gentle strokes to avoid unnecessary clouding of the PBS. Once found, ensure that the granulosa cell layer is not adherent to the yolk, as peeling granulosa from the yolk is far more difficult than separating it from the theca layer. It is suggested that the researcher first detaches the yolk from the entire follicular wall (granulosa and thecal tissue) before attempting to separate the granulosa cell layer from the theca layer. Always note that the granulosa cell layer is a very thin layer with a consistency similar to tissue paper, which can be easily torn if the researcher is not careful^8,22.

As with all methods, there are a few limitations to this protocol. The first is that the granulosa cell layer does not always come off as an intact sheet. Both patience and practice are essential to increase the likelihood of successful removal of an intact granulosa layer. Longer exposure to cold PBS may also help with this issue. Another limitation is incomplete yolk extraction, as described above. If yolk residue persists, additional cleaning steps may be required throughout DNA/RNA extraction protocols, or lipid digestion may be required prior to live cell/culture methods^23,24.

If a researcher cannot grossly visualize the granulosa layer due to a visual disability or smaller follicle size, a dissecting microscope may be used to assist in this procedure¹⁵. Use of this procedure on smaller preovulatory follicle sizes, such as the F5 follicle, may present with unique complications, such as a harder time finding the granulosa cell layering and separating it from the theca layer²². Follicle inversion, described below, may help with this issue. Upon opening the chilled follicle, the entire follicular wall may be reflected outward, resulting in the expulsion of all scant yolk contents while maintaining the integrity of the granulosa layer where it adheres upon the now-inverted theca layer. Follicular inversion should only occur after visual confirmation of the granulosa cell layer to allow for correct identification and labeling of the separated tissues. This also allows the granulosa cell layer to stay more intact as a single continuous sheet when separating it from the theca layer, particularly in smaller F5 or F6 follicles. Furthermore, to ensure that no small pieces of the granulosa cell layer are still adhered to the theca cell layer, it is important to gently agitate the inverted thecal layer in PBS. This method allows for any remnants of the granulosa cell layer to separate from the theca prior to thecal specimen storage. If this step is not taken, then it is possible that microscopic remnants of granulosa cells may remain during subsequent analyses.

It is important to note that this protocol to separate specific follicular structures of poultry can be applied across diverse research fields of study. Understanding reproductive efficiency, fertility, and reproductive disease is critical for both agricultural and biomedical fields. Proper extraction of ovarian follicular granulosa and thecal cells will lead to greater consistency and reproducibility across reproductive research regardless of the field of study, particularly considering recent advances in multi-omics analyses.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3.5 in. Small Glass Bowls, 5 oz	Amazon	B0BXP5PJTN	Autoclavable glass bowls of at least 3.5 in. diameter
Aluminum Foil	Costco	720	Cover autoclave bowls
Amazon Pet Training Pads, Regular, 100-Count	Amazon	B0B58WTPFS	For use as necrospy pads, any absorbant pad will work
Autoclave Tape	Fisherbrand	15-901-111
Cole-Parmer LED Fiber Optic Illuminators	Cole-Parmer	EW-41723-02
Curved Very Fine Precision Tip Forceps	Fisherbrand	16-100-123	Non-Serrated
Dissecting Microscope	Leica	S6E
Fine Precision Scissors	Fisherbrand	12-000-155	Non-Serrated
Food Container Boxes with Lid Set of 17 Clear/Green Microwave Freezer Dishwasher Safe	Amazon	B09QGZCRDB	Use any container that can hold ice AND an 3.5 in. small glass bowl
High Precision 45 Degree Curved Tapered Very Fine Point Tweezers/Forceps	Fisherbrand	12-000-125	Non-Serrated
High Precision Straight Very Fine Point Tweezers/Forceps	Fisherbrand	16-100-120	Non-Serrated
Isopropanol	Fisherbrand	A426P-4
McKesson Specimen Container, Sterile, Screw Cap, Leak-Resistant, 120 mL	McKesson	16-9526	4 oz. / 120 cc, graduated
Phosphate-buffered saline (PBS, 10x), pH 7.6	ThermoFisher	J62692.K7	Will need to be made into 1x PBS
Spray Bottle	Cole-Parmer	EW-06091-01	For 70% Isopropanol
Sterile Surgical Blades #22	Cincinnati Surgical	122
Sterilization Pouches 10" x 16"	Amazon	B07MFB455C
Tapered Ultrafine Tip Forceps Fisherbrand	Fisherbrand	16-100-121	Non-Serrated
Foam Biopsy Pads, Rectangular	Fisherbrand	22-038-221
Formalin Solution, 10% (Histological)	Fisher Chemical	SF98-4
Tissue processing/embedding cassettes with lid	Simport M490-2	Z672122