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JoVE Journal Biology

Biology

Isolation of Small Preantral Follicles from the Bovine Ovary Using a Combination of Fragmentation, Homogenization, and Serial Filtration

Published: September 27, 2022 doi: 10.3791/64423
Automatic Translation
1, 1, 1, 1
1Department of Animal Science, University of California Davis

Summary

Advancing the study of preantral folliculogenesis requires efficient methods of follicle isolation from single ovaries. Presented here is a streamlined, mechanical protocol for follicle isolation from bovine ovaries using a tissue chopper and homogenizer. This method allows collection of a large number of viable preantral follicles from a single ovary.

Abstract

Understanding the full process of mammalian folliculogenesis is crucial for improving assisted reproductive technologies in livestock, humans, and endangered species. Research has been mostly limited to antral and large preantral follicles due to difficulty in the isolation of smaller preantral follicles, especially in large mammals such as bovine species. This work presents an efficient approach to retrieve large numbers of small preantral follicles from a single bovine ovary. The cortex of individual bovine ovaries was sliced into 500 µm cubes using a tissue chopper and homogenized for 6 min at 9,000-11,000 rpm using a 10 mm probe. Large debris was separated from the homogenate using a cheese cloth, followed by serial filtration through 300 µm and 40 µm cell strainers. The contents retained in the 40 µm strainer were rinsed into a search dish, where follicles were identified and collected into a drop of medium. The viability of the collected follicles was tested via trypan blue staining. This method enables the isolation of a large number of viable small preantral follicles from a single bovine ovary in approximately 90 min. Importantly, this method is entirely mechanical and avoids the use of enzymes to dissociate the tissue, which may damage the follicles. The follicles obtained using this protocol can be used for downstream applications such as isolation of RNA for RT-qPCR, immunolocalization of specific proteins, and in vitro culture.

Introduction

Ovarian follicles are the functional units of the ovary, responsible for production of the gamete (oocyte) as well as hormones critical for reproductive function and overall health. Primordial follicles form in the ovary during fetal development or in the neonatal period depending on the species1, and they constitute a female's ovarian reserve. Follicular growth begins with the activation of primordial follicles that leave the resting pool and enter the growing phase. Preantral folliculogenesis, encompassing all follicle stages before antrum development, is a highly dynamic process that requires synchronous morphological and metabolic changes in the oocyte and the surrounding granulosa cells, driven by tight communication between these two cell types2,3. Preantral follicles constitute the majority of the follicular units found in the ovary at any given time4. Development through the preantral stages of folliculogenesis is estimated to be several weeks longer than antral development5,6, and this time is necessary for the oocyte and somatic cells to acquire sufficient maturity to enter the final stage of development (i.e., the antral stage), and prepare for ovulation, fertilization, and embryonic development7,8,9.

Much of the current knowledge about ovarian preantral folliculogenesis comes from mouse models10,11,12,13, due in part to the ease in recovering a large number of these follicles from a smaller and less fibrous ovary. Although reports of isolation of large numbers of preantral follicles from bovine ovaries date back approximately 30 years14, a more complete understanding about the processes regulating the development of these early-stage follicles has remained unrealized, largely due to the lack of optimized, efficient, and repeatable methods to retrieve sufficient numbers of viable preantral follicles, particularly at early stages of development. With the increasing interest in preserving the ovarian reserve for future use in assisted reproduction in humans, cows become an attractive model due to their more similar ovarian structure15. However, the bovine ovary is markedly richer in collagen compared to the mouse ovary16, making mechanical isolation using methods described for the mouse very inefficient. Efforts to expand fertility preservation techniques include complete in vitro growth of preantral follicles to the antral stage, followed by in vitro maturation (IVM) of the enclosed oocytes, in vitro fertilization (IVF), and embryo production and transfer17. Thus far, this entire process has only been achieved in mice18. In cattle, the progress toward follicle growth in vitro is limited to a few reports with variable follicle stages at the start of culture, as well as variable length of culture between protocols17,19.

The methods described in the literature for the harvest of preantral follicles from the bovine ovary have mostly used mechanical and enzymatic techniques, either isolated or in combination2,14,17,20. The first report of a protocol for bovine preantral follicle isolation used a tissue homogenizer and serial filtration to process whole ovaries20. This study was followed by reports combining mechanical and enzymatic procedures that utilized collagenase14. A recurrent theme when utilizing collagenase to digest the ovarian tissue is the potential risk for damage of the follicular basement membrane, which may compromise follicle viability14,21,22,23. Therefore, different combinations of mechanical methods have been employed, such as the use of a tissue chopper and repeated pipetting or a tissue chopper combined with homogenization20,24,25,26. Another mechanical technique that has been described utilizes needles to dissect preantral follicles directly from the ovarian tissue, which is especially useful for isolating larger (>200 µm) secondary follicles. However, this process is time-consuming, inefficient for isolating smaller preantral follicles, and is skillset-dependent when attempted in bovine ovaries19,27,28.

Taking advantage of the different techniques described in the literature, this protocol aimed to optimize the isolation of preantral follicles from single bovine ovaries in a simple, consistent, and efficient manner that avoids incubation in enzymatic solutions. Improving the methods to isolate preantral follicles will provide an opportunity to enhance the understanding of this stage of folliculogenesis and enable the development of effective culture systems to develop preantral follicles to the antral stage. The detailed procedures described herein for the isolation of preantral follicles from a large mammal such as the bovine species will be vital for researchers aiming to study early folliculogenesis in a non-murine species that is translatable to humans.

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Protocol

Bovine (Bos taurus) ovaries were sourced from a local abattoir and transported to the laboratory within 6 h of collection. Due to the large number of animals processed in the facility, the age, breed, and stage of the estrus cycle of the animals are unknown. Because no live animals were used in these experiments, an approved animal care and use protocol was not required.

1. Preparation of equipment and reagents

  1. Cover a 2 ft wide section of a lab bench with bench paper.
  2. Obtain a scalpel handle, sterile scalpel blade, hemostat, pair of dissection forceps, 20 mL luer-lock syringe, 18 G needle, two 200 mL beakers, 500 mL Erlenmeyer flask, 104 mm diameter plastic funnel, plastic cutting board, one 22 cm2 layer of cheesecloth (the cheesecloth can be sterilized by autoclaving prior to use) per ovary being processed, a 300 µm cell strainer, and a 40 µm cell strainer (see Table of Materials).
  3. Transfer all equipment onto the bench paper.
  4. Use the hemostat to slot the scalpel blade onto the scalpel handle. Align the angled base of the blade to the angled indicator on the handle, then slide the blade into the groove of the handle.
  5. Place the funnel into the Erlenmeyer flask and cover the funnel opening with the cheesecloth.
  6. Place one 50 mL conical tube per ovary to be processed in a water or bead bath set to 38.5 °C.
  7. Place one 100 mm x 15 mm square Petri dish per ovary being processed on a slide warmer set to 38.5 °C.
  8. Add 10 mL of Penicillin-Streptomycin (PenStrep; 10,000 U/mL penicillin and 10,000 µg/mL streptomycin) to 1 L of 1x phosphate buffered saline (PBS). Warm the PBS + PenStrep in a water or bead bath set to 38.5 °C at least 2 h prior to ovary processing.
    ​NOTE: PBS + PenStrep solution is imperative for washing ovaries when isolated follicles will be cultured, and it is still recommended for any downstream experiments to mitigate microbial contamination.
  9. For collecting processed ovary filtrate, use Follicle Wash Medium (FWM) consisting of TCM199 with Hank's Salts (see Table of Materials) containing 3 mg/mL bovine serum albumin (BSA), 25 mM HEPES buffer, 100 UI penicillin/100 µg/mL streptomycin, 1 mM sodium pyruvate (NaPyr), and 100 nM non-essential amino acids (NEAA).
    1. Transfer sterile TCM199, a 250 mL bottle, and a 100 mL graduated cylinder to a biosafety cabinet (BSC). Transfer 194 mL of TCM199 to the bottle.
    2. Remove the beaker of TCM199 from the BSC and bring to a stir plate. Add 600 mg of BSA, 1.19 g of HEPES buffer, and an autoclaved stir bar to the bottle and stir until dissolved.
    3. Once the BSA and HEPES buffer have completely dissolved, add 1 N sodium hydroxide (NaOH) to the medium until it reaches a pH of 7.6-7.8, as measured by a pH meter.
    4. Wipe the bottle of medium, a vacuum filtration device, four 50 mL conical tubes, and the bottles of PenStrep, NaPyr, and NEAA with 70% ethanol before transferring to the BSC.
    5. Add 2 mL each of PenStrep (10,000 U/mL penicillin and 10,000 µg/mL streptomycin), 100 mM NaPyr, and 100x NEAA to the bottle of TCM199 + 3 mg/mL BSA + 25 mM HEPES. Sterile-filter the final medium and aliquot into the 50 mL conical tubes. Store the medium at 4 °C for up to 2 weeks.
    6. Warm one 50 mL conical tube of medium per two ovaries in a bead bath set to 38.5 °C at least 1 h prior to ovary processing.

2. Tissue chopper setup

  1. Ensure the tissue chopper (see Table of Materials) is plugged in and turned on.
  2. Set the slice thickness to 500 µm, the blade force control knob to 20°, and the speed control knob to 90° according to the manufacturer's specifications.
  3. Insert a 60 mm plastic Petri dish into the plate holder and insert the plate holder onto its stage.
  4. Raise the chopping arm as high as it will go by turning the manual operating knob clockwise.
  5. Using forceps, place a double-edged razor blade (see Table of Materials) onto the screw inserted into the chopping arm. Place the blade clasp over the blade and secure with the washer and nut. Leave the nut a quarter turn loose.
  6. Rotate the manual operating knob until the chopping arm snaps the blade flat onto the Petri dish. Tighten the nut the rest of the way with the nutdriver.
  7. Raise the chopping arm as high as it will go using the manual operating knob. Move the table release knob all the way to the left until it notches into place.

3. Ovary preparation

  1. Transfer ovaries in the laboratory to warm (38.5 °C) sterile PBS + PenStrep.
    NOTE: It is recommended to process the ovaries for follicle isolation as soon as it is feasible after removal from the animal. In this protocol, ovaries were processed within 6 h of harvesting. Ovaries were transported from the abattoir to the laboratory in thermoses containing sterile 0.9% saline solution at approximately 38.5 °C.
  2. If possible, select small (≤ 4 cm x 3 cm x 3 cm) ovaries containing small (3-5 mm) antral follicles, no large (≥8 mm) antral follicles, and no prominent corpus luteum (Figure 1). These criteria are recommended to ensure that a minimal amount of non-follicle debris, such as stromal cells and extracellular matrix, is included in the resulting square dish containing isolated follicles.
    NOTE: Antral follicles can be identified as spherical, fluid-filled vesicular structures on the surface of the ovary. Corpora lutea can be identified as red, orange, or yellow stiff structures protruding from the surface of the ovary.
  3. Use scissors to remove any excess connective tissue and fat from the ovaries.
  4. Wash the ovaries for 30 s in 70% ethanol in a beaker.
  5. Wash the ovaries 3x for 2 min each in beakers of warm (38.5 °C) PBS + PenStrep, using fresh PBS + PenStrep for each wash.
  6. Keep the ovaries in warm (38.5 °C) PBS + PenStrep until ready for processing.
    NOTE: Distance between the laboratory and ovary source can be variable. Therefore, it is important to complete the protocol in a timely manner to ensure maintenance of follicular viability.

Figure 1
Figure 1: Anatomy of the bovine ovary. The bovine ovary consists of two main regions enclosed in an epithelial layer. The cortex, comprised of the tissue to the left of the dashed line, contains ovarian follicles from the primordial stage to the antral stage. Preantral follicles are too small to see with the naked eye; antral follicles are marked with asterisks. The medulla, comprised of the tissue to the right of the dashed line, contains blood vessels, lymphatic vessels, and nerves. Please click here to view a larger version of this figure.

4. Chop procedure

NOTE: Only process one ovary at a time. Process ovaries quickly to avoid temperature decreases, which may affect follicle viability.

  1. Transfer one ovary to the cutting board on the bench paper (Figure 2A) and prepare the tissue chopper (Figure 2B).
  2. Using forceps and a scalpel, cut the ovary in half and remove the medulla from each half, leaving only the cortex at a thickness of approximately 1 mm as shown in Figure 2C.
    1. Cut the ovary in half longitudinally from one ligament attachment site to the opposite attachment site.
    2. Keep one half of the ovary on the cutting board to be processed and place the other half of the ovary back into warm (38.5 °C) PBS + PenStrep.
    3. With the exposed medulla facing upward, slice along the curvature of the ovary approximately 2 mm away from the surface of the ovary without cutting through the cortex.
    4. Use the slice along the curvature of the ovary as a guide to deepen the slice, still following the curvature of the ovary to separate the cortex from the medulla.
    5. Dissect and discard any corpora lutea from the ovary by cutting along the border of the corpus luteum.
    6. Flip the ovary half over so that the epithelium faces upward and use the scalpel to finish cutting the medulla away from the cortex. Trim away any remaining white connective tissue around the edge of the ovary piece that was connected to the ligaments.
    7. Once the majority of the medulla is removed, use the scalpel to cut the cortex to approximately 1 mm thickness. Manipulate the scalpel with small, back-and-forth motions to shave away the remainder of the medulla.
      NOTE: The medulla is the inner portion of the ovary containing large blood vessels. The cortex is the outer portion of the ovary, lying directly underneath the outermost surface epithelium. The cortex is approximately 1 mm thick in the bovine ovary, and thus cutting the ovary to a thickness of 1 mm will remove the medulla.
  3. Cut the cortex into pieces no larger than 2.5 cm x 2.5 cm. Keep the cortex pieces in warm (38.5 °C) PBS + PenStrep until ready to chop.
  4. Fill a beaker with at least 50 mL of warm (38.5 °C) PBS + PenStrep and obtain a plastic transfer pipette.
  5. Transfer a single piece of cortex to the Petri dish on the tissue chopper and wet the tissue with three or four drops of warm (38.5 °C) PBS + PenStrep.
  6. Hold the piece of tissue steady with a pair of forceps and press the reset button once to start the tissue chopper. Stabilize the Petri dish with one hand while continuing to stabilize the tissue with the forceps. Move the forceps left along the tissue as needed to avoid the blade hitting the forceps. The resulting strips will be approximately 500 µm in length.
  7. Once the entire piece of cortex has been cut into strips, use the blade holder knob to lift the blade off the Petri dish and the forceps to remove any tissue from the blade.
  8. Rotate the plate holder 90°.
  9. Press the reset button once. Stabilize the Petri dish with one hand while using the forceps to push the tissue strips into the path of the blade.
  10. Pass the blade entirely through the tissue strips. Use the blade holder knob to lift the blade off the Petri dish and the forceps to remove any tissue from the blade.
  11. Use the transfer pipette and warm (38.5 °C) PBS + PenStrep to wash the chopped tissue (final size of tissue: 500 µm x 500 µm x 1 mm cubes) into a pre-warmed (38.5 °C) 50 mL conical tube. Return the conical tube to the water or bead bath to keep the chopped tissue warm (38.5 °C).
  12. Use the nutdriver to remove the nut from the chopping arm, and remove the washer and blade clasp. Using forceps, remove the blade from the chopping arm, flip it over so that the unused edge is facing the Petri dish, and place it back onto the chopping arm. Replace the blade clasp, washer, and nut, and reset the table release knob as described in steps 2.5-2.7.
  13. Repeat steps 4.5-4.12 for all remaining pieces of cortex from the ovary, replacing blades with new ones after each cutting edge has been used.
  14. Dispose of all used blades in a hard-walled, plastic sharps container.

5. Homogenization procedure

  1. Ensure the homogenizer unit (see Table of Materials) is plugged in and the speed is set to the second bar (9,000-11,000 rpm). Insert the 10 mm generator probe into the unit according to the manufacturer's specifications.
  2. Set a timer for 1 min and insert the probe into the 50 mL conical tube containing the chopped cortex tissue from one ovary (step 4.11) and enough PBS + PenStrep to fill the tube to the 25 mL line. The depth to which the probe is inserted must be 1/3 of the liquid's height measured from the bottom of the chamber. Position the probe slightly off center to minimize vortexing.
  3. Start the timer and turn on the homogenizer. Ensure the bottom of the probe does not touch the tube and hold the tube still while the homogenizer is turned on.
  4. After 1 min of homogenization, remove the probe from the tube. Using forceps, remove any connective tissue clogging the venting holes and the space between the rotor knife and rotor tube. If any pieces of cortex are stuck in the probe, remove them with forceps and place them back into the tube.
  5. Repeat steps 5.2-5.4 an additional 5x for a total of 6 min of homogenization.
  6. Place the tube with homogenized tissue into the water or bead bath to keep the tissue warm (38.5 °C). After processing the last ovary, immediately disassemble, clean, and dry the generator probe according to the manufacturer's specifications.

6. Filtration procedure

  1. Pour the dispersed tissue into the cheesecloth-covered funnel inserted into the Erlenmeyer flask. Rinse the contents of the tube into the funnel using warm (38.5 °C) PBS + PenStrep until no tissue fragments remain in the tube.
  2. Force the tissue fragments to pass through the holes of the cloth by twisting the cheesecloth around the tissue fragments and squeezing until all excess fluid and tissue are removed from the cheesecloth.
  3. Reopen the cheesecloth over the funnel, rinse the cheesecloth with PBS + PenStrep using a transfer pipette, and again squeeze any residual tissue fragments through the cloth.
  4. Use a hemostat to hold the 300 µm cell strainer over a 200 mL beaker. Pour the filtrate in the Erlenmeyer flask through the cell strainer. Rinse the contents of the flask into the cell strainer using warm (38.5 °C) PBS + PenStrep until no tissue fragments remain.
    1. If the cell strainer becomes clogged with tissue, gently tap the cell strainer against the beaker to ensure all liquid has filtered into the beaker, and then turn the cell strainer upside down and tap out the large tissue debris onto the bench paper. Return the cell strainer over the beaker and continue pouring the filtrate through it. Repeat as necessary until all filtrate from the Erlenmeyer flask has been filtered through.
  5. Use a hemostat to hold the 40 µm cell strainer over a second 200 mL beaker. Pour the filtrate in the first 200 mL beaker through the cell strainer. Rinse the contents of the beaker into the cell strainer using warm (38.5 °C) PBS + PenStrep until no tissue fragments remain. Do not discard the contents of the 40 µm cell strainer.
  6. Fit the 18 G needle to the 20 mL syringe. Fill the syringe with FWM. Turn the 40 µm cell strainer upside down over a square Petri dish and use the syringe to wash out the contents of the cell strainer into the dish. Refill the syringe and rinse the cell strainer as needed until no tissue fragments remain.
    NOTE: Typically, 25 mL of FWM is sufficient to fully rinse out the contents of the 40 µm cell strainer.

Figure 2
Figure 2: Workspace setup for ovary processing and protocol workflow. (A) Bench setup for cutting ovaries prior to chopping and for filtering the ovary homogenate. (B) Tissue chopper and homogenizer set up, with Styrofoam support to reduce vibrations of the homogenizer stage. (C) Schematic illustrating the workflow for the processing of one whole ovary. Ovaries are trimmed of excess connective tissue and then cut in half, and the medulla is removed until a ~1 mm thick slice of cortex remains. The cortex is cut into 2.5 cm x 2.5 cm pieces and chopped in a tissue chopper set to a cut interval of 500 µm. The pieces are then homogenized, and the homogenate is filtered through cheesecloth followed by filtration through 300 µm and 40 µm cell strainers. The contents of the 40 µm cell strainer are rinsed into a square Petri dish, which is searched for follicles using a stereomicroscope. Created with BioRender.com. Please click here to view a larger version of this figure.

7. Searching and collecting follicles

  1. Transfer the square Petri dish (step 6.6) to a stereoscope with a warmed stage set to 38.5 °C. The stereoscope magnification should be set between 1.25x and 3.2x depending on the searcher's preference.
  2. Pipette 10 µL drops of FWM into a 60 mm Petri dish and cover the drops with mineral oil to prevent drying out. Place the Petri dish with media drops onto a warming plate set to 38.5 °C.
    NOTE: A 4-well plate can be used to collect follicles. Add 500 µL of wash media to one or two wells. Place on the warming plate set to 38.5 °C.
  3. Obtain a micropipette plunger and tip.
    NOTE: A glass 1-5 µL micropipette (see Table of Materials) is recommended because the follicles are less likely to adhere to the glass pipette and be lost when transferred between solutions. It is also a small enough instrument to allow easier and more precise micromanipulations of the follicles.
  4. Identify follicles from the square Petri dish and transfer to media (FWM) drops using the micropipette. Many follicles are likely to be embedded in tissue debris and may be retrievable using one of two methods as described below.
    NOTE: Follicles are oblong, rather than perfect spheres, and typically have an oocyte, presenting as a solid white circle in darker contrasts, toward the center of the follicle (Figure 3A-C). Take care to avoid confusing follicles with denuded oocytes. Oocytes tend to be perfect spheres and are surrounded by a thick, clear membrane (the zona pellucida). An inverted microscope with a magnification of 10x (or greater) can be used for closer examination of follicles (Figure 3D).
    1. Carefully separate the follicles from debris using the tip of the micropipette or fine (27 G) needles.
    2. Alternatively, use a glass Pasteur pipette with a rubber bulb to take up and squirt out the debris in the dish multiple times to dislodge follicles from the debris.
  5. Work quickly, taking no longer than 30 min, to search the Petri dish to help preserve the follicle viability.
  6. Place a maximum of only five follicles per 10 µL drop, as a higher density can increase the likelihood of follicles adhering together.

8. Trypan blue exclusion viability test

NOTE: Use the lid of a Petri dish or a 4-well plate for all the following steps, as the follicles stick less to the plastic of the lid than they do to the plastic of the actual dish.

  1. Prepare PBS + 0.2% polyvinylpyrrolidone (PVP) by dissolving 100 mg PVP in 50 mL of PBS.
    NOTE: PVP is used here to reduce the likelihood of follicles attaching to the dish.
  2. Use the micropipette to transfer all follicles (average of 40) from the media drops into a 50 µL drop of PBS + 0.2% PVP.
  3. Wash the follicles 2x by transferring them sequentially to fresh 50 µL drops of PBS + 0.2% PVP.
  4. Transfer the follicles to a 285 µL drop of PBS + 0.2% PVP.
  5. Add 15 µL of trypan blue to the 285 µL drop of PBS + 0.2% PVP (final concentration of 0.05% trypan blue) and carefully mix the drop using a 200 µL pipette tip set to 100 µL.
    NOTE: If using the 4-well plate for the trypan viability test, add 475 µL of PBS + 0.2% PVP and 25 µL of trypan blue to one well.
  6. Incubate the follicles for 1 min in the trypan blue drop, and then transfer the follicles to a 50 µL drop (or 500 µL well) of PBS + 0.2% PVP.
  7. Wash the follicles 3x according to step 8.3 with fresh 50 µL drops (or 500 µL per well) of PBS + 0.2% PVP.
  8. Discard any follicles that still appear blue after three washes in PBS + 0.2% PVP, as these are non-viable. Any follicles that do not retain blue coloration after three washes are viable and may be used for immunofluorescence, culture, or other procedures (Figure 3E). Snap-freeze the follicles in liquid nitrogen and store at -80 °C until further use if required.
  9. Perform RT-qPCR analysis and immunofluorescence staining of the follicles as described in steps 9 and 10.

Figure 3
Figure 3: Isolated follicles and trypan blue exclusion test. (A-C) Isolated follicles were imaged through a stereomicroscope at several magnifications. (A) Isolated follicles among debris within the initial search dish. Individual follicles are circled in red. Scale bar = 2,000 µm. (B) Isolated follicles and debris within a droplet of follicle wash medium covered with mineral oil. Scale bar = 1,000 µm. (C) Isolated follicles without debris at a higher magnification. Scale bar = 1,000 µm. (D) Isolated follicles imaged using an inverted brightfield microscope. Scale bar = 100 µm. (E) Representative images of viable (unstained) and non-viable (blue stain) follicles imaged using an inverted brightfield microscope and a 20x objective. Scale bar = 100 µm. Please click here to view a larger version of this figure.

9. RT-qPCR analysis

  1. Isolate RNA from viable follicles (from step 8.8) using an RNA isolation reagent (see Table of Materials). Purify the RNA and treat with DNase using a commercially available cleanup kit (see Table of Materials) according to the manufacturer's instructions.
  2. Elute the RNA with 14 µL of RNase-free water and quantify using a spectrophotometer. The RNA can be stored at -80 °C until cDNA synthesis.
  3. Perform cDNA synthesis from equal amounts of RNA extracted from primary and early secondary follicles, using a commercially available cDNA synthesis kit (see Table of Materials) according to the manufacturer's instructions. Incubate the reaction mixture for 5 min at 25 °C followed by 60 min at 42 °C, then terminate the reaction by heating at 70 °C for 5 min.
  4. Perform RT-qPCR with the synthesized cDNA (5 ng per reaction) and primers (Table 1) using a commercially available reaction mix (see Table of Materials). Use thermal cycling conditions: 30 s at 95 °C for polymerase activation, followed by 40 cycles of amplification, where each cycle included 15 s at 95 °C for denaturation and 30 s at 60 °C for annealing/extension. Analyze the RT-qPCR by quantifying cycle threshold (Ct) values and/or view PCR products using agarose gel electrophoresis.
    NOTE: Transcript expression of the granulosa cell marker FSHR and germ cell marker DAZL were evaluated in this study. Reference genes were H2A and ACTB.
  5. Run a melt curve analysis by increasing the temperature from 65 °C in 0.5 °C increments every 5 s until it reaches 95 °C.

10. Immunofluorescence analysis

  1. Fix viable follicles (from step 8.8) for 15 min in a 100 µL drop of 4% (v/v) paraformaldehyde (PFA) at room temperature (RT), followed by washing 3x in 100 µL drops of PBS + 0.1% BSA + 0.1% Tween 20.
  2. Block the follicles for 1 h at RT in a blocking buffer consisting of 1x PBS + 5% (v/v) normal donkey serum (NDS). After blocking, incubate the follicles overnight at 4 °C in a 100 µL drop of 4 µg/mL rabbit anti-human CX37 antibody or 4 µg/mL rabbit isotype IgG (negative control) diluted in blocking buffer.
  3. Wash the follicles 3x in 100 µL drops of PBS + 0.1% BSA + 0.1% Tween 20, and then incubate them for 1 h at RT in the dark in a 100 µL drop of 2 µg/mL donkey-anti-rabbit AlexaFluor 488 secondary antibody diluted in blocking buffer.
  4. Incubate the follicles for 5 min at RT in the dark in a 100 µL drop of 1 µg/mL Hoechst 33342 diluted in blocking buffer to label DNA.
  5. Transfer the follicles to a 5 µL drop of mounting media (see Table of Materials) on a glass microscope slide and cover with a cover slip. Leave the slides to cure at RT overnight, followed by sealing with nail polish. Store them at 4 °C until imaging.
  6. Image all slides within 48 h of cover slipping. Perform imaging using an inverted epifluorescence microscope (see Table of Materials) under DAPI (excitation 380 nm and emission 450 nm) and FITC (excitation 470 nm and emission 525 nm) filters.
  7. Fix the exposure time for both channels. Adjust the FITC (CX37) exposure time based on the rabbit isotype negative control. Use a 20x objective and the DAPI channel set to 50 ms exposure time to identify rabbit isotype-labeled follicles.
  8. Image these follicles under the FITC channel, and decrease the exposure time until all background green signal is abolished. Note this exposure time.
  9. Image all CX37 antibody-labeled follicles using the exposure time set for the isotype FITC channel and 50 ms exposure time for the DAPI channel.
  10. Process the signal intensity, as measured by mean gray area after thresholding, using a computer image processing program29 (see Table of Materials).
    1. Adjust the tiff file of the DAPI image for each follicle such that the entire follicle is outlined. Use the Analyze Particles function of the program to select the entire follicle as a region of interest (ROI).
    2. Open the tiff file of the FITC image for the corresponding follicle and overlay the ROI generated from the DAPI image on top of the FITC image. Use the Measure function of the program to quantify the mean gray area of the FITC image, which represents signal intensity.

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Representative Results

Overview and critical steps
Using this protocol, small bovine preantral follicles can be reliably isolated from single ovaries in experimentally relevant numbers. From a total of 30 replicates, an average of 41 follicles were obtained per replicate, with a range of 11 to 135 follicles (Figure 4A). In 14 replicates, the follicles were characterized for stage of development as previously described26 by measuring the follicle diameter using a 1 µm microscope calibration slide under the stereomicroscope. Using this method, a total of 476 follicles were classified as either primary (follicles measuring 40-79 µm), early secondary (80-119 µm), or secondary (≥120 µm) follicles (Figure 4B). Primordial follicles were excluded due to the 40 µm filtration step. Follicle viability was evaluated using the trypan blue exclusion test as described previously30. Isolated follicles were briefly incubated for 1 min in 0.05% (v/v) trypan blue in PBS containing 0.2% PVP, followed by three washes with PBS + PVP and examination under a stereoscope. Using the procedure described, 100% of isolated follicles were viable in all replicates. Overall, the mechanical isolation process, from ovary collection until immediately prior to searching the Petri dish, takes approximately 30-45 min. The time spent for searching and identifying follicles takes no longer than 30 min, resulting in a process that takes a total of 1-1.25 h per ovary.

The most critical step of this protocol is ensuring that the ovarian tissue is properly homogenized. Increasing the homogenization time from 5 min to 6 min led to an increase in the number of follicles isolated (P < 0.05). An average of 41 follicles were isolated per ovary from 30 trials using a 6 min homogenization, which is a nearly threefold increase over the average of 13.8 follicles per ovary obtained from 22 trials with a 5 min homogenization time (Figure 5). Another important step is the homogenization speed, which must remain within 9,000-11,000 rpm. Higher speeds lead to decreased yields, presumably due to excessive damage and/or rupture of follicles. Follicle isolation can be further improved after the filtration steps by passing the contents of the search dish through a glass Pasteur pipette with a rubber bulb multiple times. By using the Pasteur pipette, follicles are dislodged from debris, and more follicles can be seen in the dish.

If the follicles are to be cultured after isolation, it is important to mitigate microbial contamination. Contamination can be prevented by trimming any excess connective tissue and fat from the whole ovary and then performing a series of wash steps. The trimmed ovaries should be briefly washed in 70% ethanol to reduce the microbial load, followed by several washes in warm (38.5 °C) PBS + PenStrep solution to wash away the 70% ethanol and further remove any potential microorganisms. These washes can be done without compromising follicle viability. To further prevent contamination, it is possible to perform the entire isolation protocol inside a biosafety cabinet or clean bench with sterilized equipment.

RNA isolation and RT-qPCR
Follicles of the same stage and from separate isolations were pooled, and a total of 46 primary and 34 early secondary follicles were used for analysis of mRNA expression. RNA was isolated from the pooled follicles (primary follicles = 259 ng of total RNA and early secondary follicles = 91 ng of total RNA) and used for cDNA synthesis. Transcript expression of the granulosa cell marker FSHR19 and germ cell marker DAZL in the follicles was then evaluated using RT-qPCR. As expected, both primary and early secondary follicles expressed FSHR and DAZL transcripts, confirming a previous report in human follicles31 and demonstrating that the FSHR is expressed in bovine follicles at the primary stage of development32 (Figure 6A,B).

Immunoflourescent localization of Connexin 37
Immunofluorescent localization of proteins can be performed in whole follicles. Here, this technique was used to localize the gap junction protein Connexin 37 (CX37) in isolated preantral follicles at both the primary and early secondary stages. CX37 is critical for communication between the oocyte and granulosa cells of the follicle and has been identified in both cell types at all stages of follicle development in bovine species in histological analyses33. After the trypan blue exclusion test, viable follicles were fixed in PFA and incubated with rabbit anti-human CX37 antibody or rabbit isotype IgG (negative control), followed by incubation with donkey-anti-rabbit AlexaFluor 488 secondary antibody. Hoechst 33342 was used to label DNA. The follicles were imaged using an inverted epifluorescence microscope under DAPI and FITC filters, and the signal intensity was quantified. CX37 was found to localize to the ooplasm, although notably absent from the oocyte nucleus, and to the membrane of the granulosa cells (Figure 7). These results are consistent with the known localization of CX37 to the oocyte and membrane of the granulosa cells33. The immunolocalization of proteins known to be critical for cell communication and follicle growth, such as CX37 (shown here) and FSHR32 is a vital tool to study the development of preantral follicles, effects of interventions on follicle growth, structure, and quality, as well as to assess culture effectiveness by comparing in vivo-obtained and in vitro-cultured follicles.

Gene Forward sequence (5'-3') Reverse sequence (5'-3') Product size (bp)
FSHR CCC AAC TCG ATG AGC TGA ATC T CAT AGC TAG GCA GGG AAC GG 132
DAZL GCC CAC AAA AGA AAT CTG TGG A ACT TAA GCA CTG CCC GAC TT 156
H2A GAG GAG CTG AAC AAG CTG TTG TTG TGG TGG CTC TCA GTC TTC 104
ACTB CTC TTC CAG CCT TCC TTC CT GGG CAG TGA TCT CTT TCT GC 178

Table 1: List of primers used for RT-qPCR.

Figure 4
Figure 4: Number and developmental stage of isolated preantral follicles. (A) Total number of isolated preantral follicles per replicate using 6 min tissue homogenization (n = 30 replicates). The average number of follicles is represented by the red line across the bars. (B) In 14 replicates, the developmental stage of the isolated follicles was recorded. The pie chart shows the distribution of developmental stages as a proportion of the total follicles isolated (n = 476). Please click here to view a larger version of this figure.

Figure 5
Figure 5: Comparison of 5- versus 6-min homogenization. Homogenization of the ovarian cortex for 6 min yielded a greater number of preantral follicles compared to homogenization at the same speed for 5 min (n = 22 replicates for 5 min and 30 replicates for 6 min; P < 0.01). Please click here to view a larger version of this figure.

Figure 6
Figure 6: Reverse-transcription quantitative PCR of transcripts expressed in isolated preantral follicles. Primary and early secondary preantral follicles expressed mRNA transcripts for markers of granulosa cells (FSHR), germ cells (DAZL), and reference genes (H2A and ACTB). (A) Agarose gel of PCR products for each gene with respective base pair (bp) product sizes. (B) Expression of FSHR and DAZL transcripts was quantified relative to that of the reference gene ACTB. NTC = non-template control, CT = cycle threshold. Please click here to view a larger version of this figure.

Figure 7
Figure 7: Immunofluorescent localization of CX37 in isolated preantral follicles. Representative images of CX37 immunolocalization in a primary (A) and early secondary (B) bovine preantral follicle. (C) Representative image of negative control rabbit isotype labeling in an early secondary bovine preantral follicle. Left panel: DNA labeling (DAPI). Middle panel: primary antibody rabbit anti-human CX37 labeling (FITC). Right panel: DAPI and FITC merged. Scale bars = 50 µm. (D) Average CX37 signal intensity according to follicle developmental stage (n = 21 primary and 11 early secondary follicles in two replicates).Please click here to view a larger version of this figure.

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Discussion

The present protocol details a reproducible method to retrieve early stage preantral follicles, specifically at primary and early secondary stages, from the bovine ovary. This protocol builds on previous reports20,25,30,34,35,36 and provides optimizations that result in the isolation of a meaningful number of follicles from an individual ovary. Preantral follicles isolated using this method are viable and can be used for downstream applications such as immunolocalization of proteins and RNA extraction for analysis of gene expression. Culturing of the isolated follicles was not attempted in this study. However, efforts are currently underway to achieve successful long-term culture of small bovine preantral follicles, which will be an important downstream application of this protocol that will help improve the understanding of ovarian folliculogenesis, perform toxicology tests, and devise ways to successfully manipulate follicles for fertility preservation.

Data from these experiments show that the 6 min homogenization is critical for proper dispersion of the ovarian cortical tissue and follicle release (Figure 4). Additional steps that contribute to thorough homogenization of the tissue are the initial dissection of the cortex from the medulla ensuring proper thickness of the cortex (~1 mm), trimming away of excess connective tissue, and chopping the cortex into small fragments. These steps prevent clogging of the homogenizer probe and unnecessary debris in the search plate. Cell strainer pore size is important for excluding debris and follicles of undesired size and stage; in the case of this protocol, the final step is collecting the contents of a 40 µm cell strainer so as to exclude primordial follicles, which are typically <40 µm in diameter in cattle26. Finally, using a Pasteur pipette to dislodge follicles from debris in the search dish is recommended over attempting to manually dissect follicles out of the debris, as the latter technique requires more technical skill and, if done improperly, can damage the follicles.

While this protocol presents improvements compared to existing methods, there are limitations that must be considered. First, due to the heterogeneous nature of the ovarian follicle population15 and unknown history of the animals from which ovaries were retrieved, the number of follicles isolated after performing this protocol were highly variable. One factor that must be considered is animal age, as the ovarian reserve is known to decrease with age37.

Isolation of small preantral follicles from the bovine ovary provides opportunities to investigate basic scientific questions at the single-sample level, an approach that has been difficult to conduct previously. For example, all preantral follicle stages evaluated here showed expression of CX37, a critical protein for oocyte-granulosa cell communication. Similarly, expression of FSH receptor has been previously demonstrated in individually isolated preantral follicles32. Other specific markers of granulosa cells, such as the steroidogenic enzymes CYP17 and CYP1938, would be relevant additions to the gene/protein expression panel of preantral follicles, and will be the subject of future projects. The visualization of the entire follicular structure via regular or confocal microscopy is more comprehensive compared to histological examination, where only one segment of the follicle can be analyzed at a time. This is especially advantageous in studies that may want to utilize immunostaining or in situ hybridization, where detailed spatial localization of expression is required. Additionally, isolated preantral follicles can be used to investigate single-follicle gene expression by using sensitive kits for RT-qPCR30. The ability to analyze follicles individually and without influence of the ovarian environment is a critical step forward in understanding the complexity of preantral folliculogenesis.

Cryopreservation of the ovarian cortex tissue has become a promising technique for safeguarding fertility in female mammals39, including women40. The preservation and restoration of fertility using preantral follicles presents an attractive assisted reproductive technology as well as a promising method for studying folliculogenesis41. Following freezing and thawing, the tissue can be subjected to this isolation protocol for retrieval of viable preantral follicles. Although the potential ability of this protocol to retrieve follicles from previously cryopreserved tissue was not specifically assessed, others have demonstrated success and used viable small preantral follicles isolated from frozen-thawed tissue for in vitro culture42,43,44,45. Furthermore, freshly isolated preantral follicles can be individually cryopreserved via vitrification and maintain structural integrity36.

An update on details for the efficient isolation of preantral follicles from a large mammalian species such as the bovine has been missing in the literature. Moreover, a visual and explicit guide on how to isolate bovine preantral follicles is much needed, as the lack of reproducibility has been a bottleneck in the field. Recently, Bus et al.36 described the isolation of bovine preantral follicles using a mechanical method, with a calculated average yield of 6.1 primary follicles per ovary. The mechanical isolation method described here results in the isolation of an average of 41 preantral follicles per ovary, the majority being at the primary stage. Therefore, this technique is especially useful for studies focused on primary follicle physiology and development. Primary follicles represent the first stage of the growing pool and are abundant in the ovarian cortex. Therefore, the use of the described technique, which is optimized to harvest this developmental stage, is novel and may be particularly desirable to take advantage of the ovarian reserve while avoiding the more difficult manipulation of primordial follicles. Another advantage of isolating primary follicles would be to associate ovarian tissue culture for the initial activation of primordial follicles with subsequent isolation of primary follicles for further culture. Given the interest in culturing follicles from the more abundant primary stage, the method presented here can incentivize researchers to pursue avenues they may have previously avoided due to the challenge of isolating primary follicles.

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Disclosures

The authors have nothing to disclose.

Acknowledgments

This project was partially funded by USDA Multi-state project W4112 and UC Davis Jastro Shields award to SM.

The authors would like to extend their appreciation to Central Valley Meat, Inc. for providing the bovine ovaries used in all experiments. The authors also thank Olivia Silvera for assistance with ovary processing and follicle isolation.

Materials

Name Company Catalog Number Comments
5-3/4" Soda Lime Disposable Glass Pasteur Pipette Duran Wheaton Kimble 63A54 Pasteur pipette that can be used to dislodge follicles from debris while searching within the petri dish
16% Paraformaldehyde Electron Microscopy Sciences 15710 Diluted to 4%; fixation of follicles for immunostaining
20 mL Luer-lock Syringe Fisher Scientific Z116882-100EA Syringe used with the 18 G needle to dislodge follicles from the 40 μm cell strainer
#21 Sterile Scalpel Blade Fisher Scientific 50-365-023 Used to cut the ovaries and remove the medula
40 μm Cell Strainer Fisher Scientific  22-363-547 Used to filter the filtrate from the 300 μm cell strainer
104 mm Plastic Funnel Fisher Scientific 10-348C Size can vary, but ensure the cheese cloth is cut appropriately and that the ovarian homogenate will not spill over
300 μm Cell Strainer pluriSelect  43-50300-03 Used to filter the filtrate from the cheese cloth 
500 mL Erlenmeyer Flask Fisher Scientific FB500500 Funnel and flask used to catch filtrate from the cheese cloth 
Air-Tite Sterile Needles 18 G Thermo Fisher Scientific 14-817-151 18 G offers enough pressure to dislodge follicles from the 40 μm cell strainer
Air-Tite Sterile Needles 27 G 13 mm Fisher Scientific 14-817-171 Needles that can be used to manipulate any debris in which follicles are stuck
BD Hoechst 33342 Solution Fisher Scientific BDB561908 Fluorescent DNA stain
Bovine Serum Albumin (BSA) Sigma-Aldrich A7030-100G  Component of follicle wash media
Cheese Cloth Electron Microscopy Sciences 71748-00 First filtering step of the ovarian homogenate meant to remove large tissue debris
Classic Double Edge Safety Razor Blades Wilkinson Sword N/A Razor blades that fit the best in the McIlwain Tissue Chopper and do not dull quickly
Donkey-Anti-Rabbit Secondary Antibody, Alexa Fluor 488 Fisher Scientific A-21206 Secondary antibody for immunostaining
Eisco Latex Pipette Bulbs Fisher Scientific S29388 Rubber bulb to use with Pasteur pipettes
HEPES Buffer Sigma-Aldrich H3375 Component of follicle wash media
Homogenizer VWR 10032-336 Homogenize the ovarian tissue to release follicles 
ImageJ/Fiji NIH v2.3.1 Software used for analysis of fluorescence-immunolocalization
McIlwain Tissue Chopper Ted Pella 10184 Used to cut ovarian tissue small enough for homogenization
Microscope - Stereoscope Olympus SZX2-ILLT Dissection microscope used for searching and harvesting follicles from the filtrate
Microscope - Inverted Nikon Diaphot 300 Inverted microscope used for high magnification brightfield visualization of isolated follicles
Microscope - Inverted ECHO Revolve R4 Inverted microscope used for high magnification brightfield and epifluorescence visualization of isolated follicles
Mineral Oil Sigma-Aldrich M8410-1L Oil to cover the drops of follicle wash medium to prevent evaporation during searching
Non-essential Amino Acids (NEAA) Gibco 11140-050 Component of follicle wash medium
Normal Donkey Serum Jackson ImmunoResearch 017-000-001 Reagent for immunostaining blocking buffer
Nunc 4-well Dishes for IVF Thermo Fisher Scientific 144444 4-well dishes for follicle isolation and washing
Penicillin-Streptomycin Solution 100x Gibco 15-140-122 Component of follicle wash medium
Petri Dish 60 mm OD x 13.7 mm Ted Pella 10184-04 Petri dish that fits the best in the McIlwain Tissue Chopper
Phosphate Buffered Saline (PBS) Fisher Scientific BP665-1 Washing buffer for ovaries and follicles
Plastic Cutting Board Fisher Scientific 09-002-24A Cutting board of sufficient size to safely cut ovaries
Polyvinylpyrrolidone (PVP) Fisher Scientific BP431-100 Addition of PVP (0.1% w/v) to PBS prevents follicles from sticking to the plate or each other 
ProLong Gold Antifade Mountant Thermo Fisher Scientific P36930 Mounting medium for fluorescently labeled cells or tissue
Qiagen RNeasy Micro Kit Qiagen 74004 RNA column clean-up kit
R The R Foundation v4.1.2 Statistical analysis software
Rabbit-Anti-Human Cx37/GJA4 Polyclonal Antibody Abcam ab181701 Cx37 primary antibody for immunostaining
RevertAid RT Reverse Transcription Kit Thermo Fisher Scientific K1691 cDNA synthesis kit
Rstudio RStudio, PBC v2021.09.2 Statistical analysis software
Sodium Hydroxide Solution (1N/Certified) Fisher Scientific SS266-1 Used to increase media pH to 7.6-7.8
Sodium Pyruvate (NaPyr) Gibco 11360-070 Component of follicle wash medium
Square Petri Dish 100 mm x 15 mm  Thermo Fisher Scientific 60872-310 Gridded petri dishes allow for more efficient identification of follicles 
SsoAdvanced Universal SYBR Green Supermix BioRad 1725271 Mastermix for PCR reaction
Steritop Threaded Bottle Top Filter Sigma-Aldrich S2GPT02RE Used to sterilize follicle wash medium
SYBR-safe DNA gel stain Thermo Fisher Scientific S33102 Staining to visual PCR products on agarose gel
TCM199 with Hank’s Salts Gibco 12-350-039 Component of follicle wash medium
Triton X-100 Fisher Scientific BP151-100 Detergent for immunostaining permeabilization buffer
Trizol reagent Thermo Fisher Scientific 15596026 RNA isolation reagent
Trypan Blue Solution, 0.4% Gibco 15-250-061 Used for testing viability of isolated follicles
Tween 20 Detergent for immunostaining wash buffer
Warmer Plate Universal WTA 20931 Warm plate to keep follicles at 38.5 °C while searching under the microscope
Wiretrol II Calibrated Micropipets Drummond 50002-005 Glass micropipettes to manipulate follicles

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References

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Tags

Isolation Small Preantral Follicles Bovine Ovary Fragmentation Homogenization Serial Filtration Folliculogenesis In Vivo Treatments Ovarian Biopsy Cryo-preservation Fertility Preservation Gonadotoxic Treatment Chemotherapy Germplasm Preservation Endangered Species Procedure Stephanie McDonnell Amanda Morton Juliana Candelaria PhD Students Laboratory Ovaries Cutting Board Scalpel
Isolation of Small Preantral Follicles from the Bovine Ovary Using a Combination of Fragmentation, Homogenization, and Serial Filtration
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McDonnell, S. P., Candelaria, J. I., … More

McDonnell, S. P., Candelaria, J. I., Morton, A. J., Denicol, A. C. Isolation of Small Preantral Follicles from the Bovine Ovary Using a Combination of Fragmentation, Homogenization, and Serial Filtration. J. Vis. Exp. (187), e64423, doi:10.3791/64423 (2022).

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