Summary
In vitro culture of bovine ovarian cortex and the effect of nutritional Stair-step diet on ovarian microenvironment is presented. Ovarian cortex pieces were cultured for seven days and steroids, cytokines, and follicle stages were evaluated. The Stair-Step diet treatment had increased steroidogenesis resulting in follicle progression in culture.
Abstract
Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors from somatic cells and cumulus cell-oocyte communication. Little is known about the ovarian microenvironment and how the cytokines and steroids produced in the surrounding milieu affect follicle progression or arrest. In vitro culture of ovarian cortex enables follicles to develop in a normalized environment that remains supported by adjacent stroma. Our objective was to determine the effect of nutritional Stair-Step diet on the ovarian microenvironment (follicle development, steroid, and cytokine production) through in vitro culture of bovine ovarian cortex. To accomplish this, ovarian cortical pieces were removed from heifers undergoing two different nutritionally developed schemes prior to puberty: Control (traditional nutrition development) and Stair-Step (feeding and restriction during development) that were cut into approximately 0.5-1 mm3 pieces. These pieces were subsequently passed through a series of washes and positioned on a tissue culture insert that is set into a well containing Waymouth's culture medium. Ovarian cortex was cultured for 7 days with daily culture media changes. Histological sectioning was performed to determine follicle stage changes before and after the culture to determine effects of nutrition and impact of culture without additional treatment. Cortex culture medium was pooled over days to measure steroids, steroid metabolites, and cytokines. There were tendencies for increased steroid hormones in ovarian microenvironment that allowed for follicle progression in the Stair-Step versus Control ovarian cortex cultures. The ovarian cortex culture technique allows for a better understanding of the ovarian microenvironment, and how alterations in endocrine secretion may affect follicle progression and growth from both in vivo and in vitro treatments. This culture method may also prove beneficial for testing potential therapeutics that may improve follicle progression in women to promote fertility.
Introduction
The ovarian cortex represents the outer layer of the ovary where follicle development occurs1. Primordial follicles, initially arrested in development, will be activated to become primary, secondary, and then antral or tertiary follicles based on paracrine and gonadotropin inputs1,2,3,4. To better understand physiological processes within the ovary, tissue culture can be used as an in vitro model, thereby allowing for a controlled environment to conduct experiments. Many studies have utilized ovarian tissue culture for research in assisted reproductive technology, fertility preservation, and ovarian cancer5,6,7. Ovarian tissue culture has also served as a model for investigating reproductive toxins that damage the ovarian health and the etiology of reproductive disorders such as Polycystic Ovary Syndrome (PCOS)8,9,10,11. Thus, this culture system is applicable to a wide array of specialties.
In rodents, whole fetal or perinatal gonads have been used in reproductive biology experiments12,13,14,15. However, gonads from larger domestic livestock cannot be cultured as whole organs due to their large size and potential degeneration. Therefore, bovine, and non-human primate ovarian cortex is cut into smaller pieces16,17,18. Many studies have cultured small ovarian cortex pieces to study various growth factor(s) in primordial follicle initiation in domestic livestock and non-human primates1,17,18,19. The use of ovarian cortex culture has also demonstrated primordial follicle initiation in the absence of serum for bovine and primate cortical pieces cultured for 7 days20. Yang and Fortune in 2006 treated fetal ovarian cortex culture medium with a range of testosterone doses over 10 days and observed that the 10-7 M concentration of testosterone increased follicle recruitment, survival, and increased progression of early stage follicles19. In 2007, using ovarian cortex cultures from bovine fetuses (5-8 months of gestation), Yang and Fortune reported a role for Vascular Endothelial Growth Factor A (VEGFA) in the primary to secondary follicle transition21. Furthermore, our laboratory has utilized ovarian cortex cultures to demonstrate how VEGFA isoforms (angiogenic, antiangiogenic, and a combination) may regulate different signal transduction pathways through the Kinase domain receptor (KDR), which is the main signal transduction receptor that VEGFA binds16. This information allowed for a better understanding of how different VEGFA isoforms affect signaling pathways to elicit follicle progression or arrest. Taken together, culturing of ovarian cortex pieces in vitro with different steroids or growth factors can be a valuable assay to determine effects on mechanisms regulating folliculogenesis. Similarly, animals that are developed on different nutritional regimes may have altered ovarian microenvironments, which may promote or inhibit folliculogenesis affecting female reproductive maturity. Thus, our goal in the current manuscript is to report the bovine cortex culture technique and determine whether there are differences in ovarian microenvironments after in vitro culture of bovine cortex from heifers fed either Control or Stair-Step diets collected at 13 months of age as described previously16.
Therefore, our next step was to determine the ovarian microenvironment in these heifers that were developed with different nutritional diets. We evaluated ovarian cortex from heifers fed with either a Stair-Step or Control diet. Controls heifers were offered a maintenance diet of 97.9 g/kg0.75 for 84 days. The Stair-Step diet was initiated at 8 months containing a restricted fed diet of 67.4 g/kg0.75 for 84 days. After the first 84 days, while Control heifers continued to receive 97.9 g/kg0.75, the Stair-Step beef heifers were offered 118.9 g/kg0.75 for another 68 days, after which they were ovariectomized at 13 months of age16 for studying changes in follicular stages and morphology before and after culture. We also assayed for differences in steroids, steroid metabolites, chemokines, and cytokines secreted into cortex media. Steroids and other metabolites were measured to determine if there were any direct effects from treatments conducted in vivo and/or in vitro on tissue viability and productivity. Changes in the ovarian microenvironment prior to and after culture provided a snapshot of the endocrine milieu and folliculogenesis prior to culture and how culture or treatment during culture affects follicle progression or arrest.
Ovaries were collected after ovariectomies were performed at the U.S. Meat Animal Research Center (USMARC) according to their IACUC procedures from Control and Stair-Step heifers at 13 months of age16, cleaned with sterile phosphate buffer saline (PBS) washes with 0.1% antibiotic to remove blood and other contaminants, trimmed excess tissue, and transported to the University of Nebraska-Lincoln (UNL) Reproductive Physiology laboratory UNL at 37°C23. At UNL, ovarian cortex pieces were cut into small square pieces (~0.5-1 mm3; Figure 1) and cultured for 7 days (Figure 2). Histology was conducted on the cortex culture slides prior to and after culture to determine follicles stages16,24 (Figure 3 and Figure 4), and extracellular matrix proteins that may indicate fibrosis (Picro-Sirus Red, PSR; Figure 5). This allowed determination of effect of in vivo nutritional regimes on follicle stages and allowed comparison of 7 days of ovarian cortex on follicle stages and follicle progression. Throughout the culture, the medium was collected and changed daily (approximately 70% of media was collected each day; 250 µL/well) so that either daily hormones/cytokines/chemokines can be assessed or pooled over days to obtain average concentrations. Steroids such as androstenedione (A4) and estrogen (E2) can be pooled over 3 days and assessed through radioimmunoassay (RIA; Figure 6) and pooled over 4 days per animal and assayed via High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS)24,25 (Table 1). Cytokine arrays were utilized to assess cytokine and chemokine concentrations in ovarian cortex culture medium26 (Table 2). Real-time polymerase chain reaction (RT-PCR) assay plates were conducted to determine gene expression for specific signal transduction pathways as demonstrated previously16. All of the steroid, cytokine, follicle stage and histological markers provide a snapshot of the ovarian microenvironment and clues as to the ability of that microenvironment to promote "normal" or "abnormal" folliculogenesis.
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Protocol
The ovaries were obtained from U. S. Meat Animal Research Center16. As stated previously16, all procedures were approved by the U.S. Meat Animal Research Center (USMARC) Animal Care and Use Committee in accordance with the guide for Care and Use of Agricultural Animals in Agricultural Research and Teaching. The ovaries were brought to the University of Nebraska-Lincoln Reproductive Laboratory where they were processed and cultured.
1. Preparation of required media
- Waymouth MB 752/1 medium
- Fill a 1 L tissue culture bottle with 900 mL of sterile water. While the water is gently stirring on a stir plate, gradually add the powdered medium. Once the powdered medium is dissolved, add 2.24 g of sodium bicarbonate followed by 1.25 g of bovine serum albumin (BSA). Use a pH meter and adjust the pH to 7.25-7.35. Add additional sterile water to bring the final volume to 1 L.
- Move to a biological safety cabinet and add penicillin-streptomycin sulfate at a concentration of 0.1% v/v of the medium. Filter the medium with a 0.22 µm pore 33.2 cm2 500 mL bottle top filter.
- Pour off the filtered medium into several 50 mL conical tubes. Add 0.5 mL of Insulin-Transferrin-Selenium per 50 mL of aliquoted medium.
- Wrap the conical tubes and stock bottle of the medium in aluminum foil and store at 4 °C. This medium is light sensitive.
NOTE: Waymouth medium can be stored for up to 1 month.
- Leibovitz's L-15 (LB-15) medium
NOTE: LB-15 medium is used to clean tissue in preparation for culture.- Fill a 1 L tissue culture bottle with 900 mL of sterile water. While the sterile water is gently stirring on a stir plate, gradually add the prepared powdered medium. Use a pH meter and adjust the pH to 7.25-7.35. Add additional sterile water to bring the final volume to 1 L.
- Move to biological safety cabinet. Make 1 L of LB-15 with 0.1% antibiotic (see Table of Materials). Filter the medium into two 500 mL tissue culture bottles using a 0.22 µm pore 33.2 cm2 500 mL bottle top filter. Wrap bottles in aluminum foil as LB-15 medium is light-sensitive and store at 4 °C.
NOTE: LB-15 medium can be stored for up to 1 month.
- Phosphate Buffered Saline (PBS)
- Make PBS in the lab or purchase sterile PBS without calcium or magnesium (Table of Materials). To make PBS in the lab, begin with 800 mL of distilled water and add 8 g of sodium chloride (NaCl) to it. Then, add 0.2 g of potassium chloride (KCl), 1.44 g of sodium phosphate dibasic (Na2HPO4), and 0.24 g of potassium phosphate dibasic (KH2PO4). Adjust pH to ~7.4 and adjust total volume to 1 L. Sterilize the solution by autoclaving.
- Make 1 L PBS with 0.1% antibiotic (see Table of Materials) while in a biological safety cabinet.
2. Ovarian cortical culture protocol
NOTE: Ovaries were obtained from spring born USMARC heifers at 13 months of age. Ovaries were rinsed thoroughly, and all blood and other fluid were removed with PBS containing antibiotic (0.1%) and transported at 37 °C23 to University of Nebraska-Lincoln Reproduction Laboratory UNL (1.5 h away). (For comments on temperature of ovaries during transport please see Discussion)
- Prepare the ovarian tissue on a clean bench (Figure 1).
- Disinfect the clean bench with 70% ethanol. Place a fresh absorbent pad on the benchtop. Ensure that the clean bench blower is turned on half an hour prior to dissection along with UV light to sterilize anything in the clean bench, including absorbent pad and make sure appropriate PPE is used.
- Arrange the Petri dishes (60 x 15 mm) for tissue washes. Three Petri dishes are required for PBS wash, three for PBS with antibiotic, and three for LB-15 washes. An additional LB-15-containing Petri dish with accompanying lid will be utilized for final placement of pieces after washes.
- Fill each Petri dish with approximately 10 mL of appropriate fluids, either PBS or LB-15.
Figure 1: Layout of plates for washing the ovary and cortex pieces in the clean bench. (A) PBS used for washing the ovary as sections of the cortex are removed. (B) PBS with antibiotic washes that cortex pieces are moved through. (C) Ovarian cortex pieces are washed four times in LB-15 before moving to the biosafety cabinet for final wash in LB-15. Please click here to view a larger version of this figure.
- Remove the prepared Waymouth and LB-15 medium from the refrigerator and warm to room temperature.
- Autoclave all tools to ensure sterilization prior to use.
- Maintain ovaries at 37 °C until ovarian cortex is ready to be collected.
- Using forceps with serrated jaws, pick up the ovary and thoroughly wash in the first PBS-filled Petri dish. Transfer the ovary to the second PBS wash and thoroughly cleanse once more.
NOTE: The ovary will stay in the second PBS wash while ovarian cortical strips are removed. - Using serrated jaw forceps, secure the ovary and slice in half. At this time, the ovarian cortex will cut away from the medulla. Using a ruler, make sure that no more than 1–2 mm of depth of surface of ovary is removed away from the medulla16. Remove transverse sections of the ovarian cortex from medulla, cut 3–4 thin strips of ovarian cortex (Figure 2) with a scalpel (#11 scalpel blade; #3 handle), and place the strips in the third PBS-filled Petri dish.
NOTE: At this time, additional ovarian cortical tissue can be collected for RNA extraction or fixed and collected for histology of initial non-cultured cortex pieces. When removing strips of ovarian cortex, avoid areas with visible antral follicles or corpora lutea. In addition, avoid collecting medullary tissue. The histology of the medulla is very different as shown previously16. If the ovarian cortex is not cut to more than a 1–2 mm depth, then the medulla should not be obtained. Distinct histology allows for landmarks between the cortex and the medulla. - Cut the ovarian cortex strips in the third PBS wash into small, square pieces (~0.5–1 mm3) with a #21 scalpel blade. Use a ruler underneath the Petri dishes to ensure the pieces are of similar size and thickness to make consistent ovarian cortex pieces. Use forceps to secure the strips while cutting the pieces with a scalpel.
NOTE: The number of tissue pieces cut is dependent on the experiment. Four pieces of ovarian cortex is the minimum amount of tissue necessary for culture. Other methods for ensuring appropriate length and depth include using special slicers26 or precut plastic pieces as templates27. - Wash ovarian cortical pieces through all three PBS with antibiotic-filled Petri dishes. Use a curved tip forceps to move pieces between washes.
- Move cortex pieces through the series of LB-15 washes and place in final LB-15-filled Petri dish. Label the lid with animal ID and ovary side (left or right).
NOTE: Fully submerge the ovarian cortex pieces in each wash for thorough cleaning. - Collect four ovarian cortex pieces per ovary and fix for day zero histology. Additional pieces can also be flash frozen for RNA. The remaining tissue pieces will be used for culture. Wipe down dissecting tools with 70% ethanol after each tissue collection.
- Prepare a biological safety cabinet for final tissue wash and culture preparation. Sanitize supplies with 70% ethanol before placing in the biological safety cabinet. Use the aseptic technique when working in the biological safety cabinet.
- Move all ovarian cortex intended for culture to the biological safety cabinet and wash once more in an LB-15-filled Petri dish.
- In a 24-well tissue culture plate, pipette 350 µL of Waymouth medium per well.
- Place uncoated culture well inserts into each well using forceps. Ensure that no bubbles are formed under the base of the insert as this would result in the tissue drying out. The medium must be touching the inserts to allow for the medium to be absorbed up and surround the ovarian cortex pieces.
- Carefully position four ovarian cortex pieces onto the mesh of each insert (Figure 2). The forceps can puncture the mesh if the tissue pieces are not delicately placed. The tissue pieces should not be touching each other or the side of the insert.
- Incubate the tissue at 37 °C with 5% CO216.
NOTE: Others have used 38.8 °C28. However, no difference has been observed in the integrity of the tissue nor in the ability of follicles to progress in 37 °C tissue nor have others39,30. Thus, at this point any of these temperatures should be conducive to experiment success. Others have used 400 µL of medium. Either amount is fine as long as one is consistent, and the tissue is partially submerged allowing for adequate surface tension to allow for hydration of tissue (media surrounding ovarian cortex pieces). Fill empty wells with 500 µL of sterile water to help reduce evaporation from other wells.
Figure 2: Ovarian cortex pieces and culture plate. (A) An ovarian strip being cut from the cortex of the ovary. (B) Ruler and cortex piece shown side by side. (C) Four cortex pieces (~0.5-1 mm3) resting on the insert in the culture medium in the plate. (D) Lifting the insert to collect the culture medium from the well. Collect and replace all the culture medium daily (250 µL) to maintain proper pH.Approximately 250 µL is obtained from each well each day (about 70% of the initial culture medium). Please click here to view a larger version of this figure.
3. Media collection
- Change ovarian cortex culture medium daily for 7 days. Medium changes should be as close to 24 h apart as possible to prevent large pH and color changes in medium. Warm Waymouth medium to 37 °C prior to medium change. Approximately 250 µL is obtained from each well each day (about 70% of initial culture medium).
- During medium changes, use forceps to gently lift the insert out of the well. Collect the cultured Waymouth medium in 0.5 mL tubes (approximately 250 µL /day). Set the insert back in well and add 350 µL of fresh culture medium by dispensing the medium between the side of the insert and well.
NOTE: Change most of the media daily to obtain enough media to measure all the steroids, cytokines, and chemokines necessary to determine ovarian microenvironment. Also, daily medium changes are important to prevent large pH changes (indicated by color change) in the medium. Drops of medium were retained surrounding the ovarian cortex pieces to ensure the pieces remained wet. No problems were observed with cultured tissue due to changing 70% of the media. - Store the collected medium from tissue culture at -20 °C.
4. Imaging and downstream processing
- After 7 days of culture at 37 °C with 5% CO2, image the ovarian cortex pieces using a dissection microscope with an attached camera and a computer imaging software program.
NOTE: A dark room is usually best for achieving the best picture quality for imaging. - After imaging, fix two ovarian cortex pieces per well in Bouins for histology and flash freeze two ovarian cortex pieces in liquid nitrogen to obtain RNA for cDNA. Repeat this step for all the wells with tissue. Collect the medium from day 7 and store at -20 °C.
- Let the ovarian cortex pieces remain immersed in Bouins (picric acid 750 mL, glacial acetic acid 50 mL, and 37%–40% formalin 250 mL) for approximately 1.5 h before being washed with 70% ethanol three times. The tissue will remain in 70% ethanol and be cleared daily until the solution is no longer yellow.
NOTE: Fixatives other than Bouins as well as paraformaldehyde can be used. In this experiement Bouins is used as it is the fixative to achieve optimal morphology. If more tissue is required for other analysis, additional wells of media and pieces of ovarian cortex can be obtained from each animal.
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Representative Results
This bovine cortex culture procedure can be used to determine a wide variety of hormone, cytokine, and histology data from small pieces of the ovary. Staining, such as hematoxylin and eosin (H&E), can be used to determine ovarian morphology through follicle staging16,23,31 (Figure 3). Briefly, follicles were classified as primordial, which is an oocyte surrounded by a single layer of squamous pre-granulosa cells (0); transitional follicle or early primary, which is an oocyte surrounded by mostly squamous pre-granulosa cells and some cuboidal granulosa cells (1); primary follicle, which is an oocyte surrounded by 1-1.5 layers of cuboidal granulosa cells (2); secondary follicle, which is an oocyte surrounded by two or more cuboidal granulosa cells (3); antral follicle, which is no larger than 1 mm in diameter and surrounded by two or more layers of granulosa cells containing a distinct antrum (4)16,23 (Figure 3). Follicle staging can be conducted on ovarian cortex fixed prior to and following culture to assess folliculogenesis (Figure 4). We took three images per slide from three different slides stained with H&E. Then, the follicles were staged and counted by three individuals and averaged to determine the number of follicles at each stage16,23. The area of the field of view for an image (three per slide) at 400x magnification is 0.4mm2. Thus, 30% of the area of the ovarian cortex pieces were counted to determine follicle stages. The initial folliclenumber (before culture) (Figure 4A) is used to normalize the follicles counted after culture (Figure 4B).
Additionally, differences in morphology as determined by collagen deposition (Picro Sirus Red staining) can indicate fibrosis in ovarian cortex from Stair Step or Control heifers (Figure 5). Daily collection of culture medium can be pooled over 3 days to assess varied steroid hormone production by RIA (using 200 µL medium sample per animal; Figure 6) or steroid metabolites using high performance liquid chromatography mass spectrometry (HPLC-MS; 220 µL medium sample pooled over 4 days per animal; Table 1) and cytokine production (Table 2). Therefore, several replicates of one animal may be required to ensure enough cortex medium to perform all the desired assays.
Because Stair-Step heifers have increased primordial follicles at the beginning of the culture we expected these to progress in culture and obtain greater number of secondary follicles, which we observed in the results. Also, due to increase in secondary follicles, we would expect greater concentrations of steroids. We did see tendency for increases in androgens, glucocorticoid metabolites, and progesterone metabolites, which would support this in the current manuscript. Our lab has also evaluated effects of different VEGFA isoforms on follicle progression, steroidogenesis, and activation of different signal transduction molecules in the KDR (also known as Vascular Endothelial Growth Factor Receptor 2; VEGFR2) using signal transduction array plates16. To reduce animal variation, we use 4-6 animals per treatment and for other experiments depending on power analysis and variability we have used as many as 11.
Repeatability of results from bovine ovarian cortex culture is most affected by contamination of ovarian cortex pieces. Additionally, negative, or subpar results can occur if the medium is not changed regularly within a 24-h period. The medium when originally added is pink in color, but when the medium is collected, and the color appears to be orange or bright yellow this could indicate a change in pH that could be detrimental to the tissue. Also, tissue pieces that are cut too large might develop degeneration in the middle that would not be observed until tissue sectioning for histology. This degeneration will limit the use of the tissue for analysis. The data presented in this paper has been analyzed using nonparametric tests and a general linear model analysis in a statistical software program. The number of primordial, primary, secondary, and antral follicles per section before and after culture were analyzed using a generalized linear mixed model. Significance was determined at P < 0.05 and a tendency was reported at 0.14 ≤ P ≥ 0.05.
Figure 3: Hematoxylin and Eosin staining for follicle staging of ovarian cortex. Different stages of follicles are indicated by arrows. (A) Primordial follicles (stage 0); (B) Early primary follicles (stage 1); (C) Primary follicles (stage 2); (D) Secondary follicle (stage 3); (E) Antral follicle (stage 4). Area of the field of view for an image at 400x magnification is 0.4 mm2. We count 30% of the area of the ovarian cortex pieces to determine the follicle stages. Please click here to view a larger version of this figure.
Figure 4: Average number of follicles at different follicular stages in Control (n=6) and Stair-Step (n=6) heifers. (A) Before culture Primordial P = 0.001, Early Primary P = 0.12, Primary P = 0.31, Secondary P = 0.22. (B) After 7 days of culture, Primordial P = 0.37, Early Primary P = 0.84, Primary P = 0.69, Secondary P = 0.02. Error bars are representative of SEM. Please click here to view a larger version of this figure.
Figure 5: Collagen (Picro Sirius Red; PSR) staining in ovarian cortex. PSR in (A) Control and Stair-Step heifers from Day 0 and Day 7. (B) Graph comparing the average area of PSR-positive staining per ovarian cortex field (pixels/µm2) between Control (n = 4) and Stair-Step (n = 4) heifers. Error bars are representative of SEM. Area of the field of view for an image at 400x magnification is 0.4mm2. Please click here to view a larger version of this figure.
Figure 6: Concentrations of A4 and E2. (A) Concentration of A4 and (B) concentration of E2 pooled over 3 days of culture in ovarian cortex media of Control and Stair-Step heifers as measured by RIA's. n = 4 for each group. Error bars are representative of SEM. Please click here to view a larger version of this figure.
| Hormones | Control | Stair-Step | P-Value | |||
| n | 4 | 4 | ||||
| ng/mL | Mean | SEM ± | Mean | SEM ± | ||
| DOC | 0.33 | 0.28 | 0.72 | 0.48 | 0.15 | |
| INN | 0.61 | 0.60 | 4.93 | 3.99 | 0.08 | |
| CORT | 0.003 | 0.003 | 0.01 | 0.01 | 0.85 | |
| 17OHP | 0.59 | 0.53 | 1.88 | 0.99 | 0.08 | |
| A4 | 1.46 | 1.43 | 5.74 | 3.27 | 0.08 | |
| AN | 0.15 | 0.09 | 0.54 | 0.32 | 0.08 | |
| DHEAS | 4.50 | 2.60 | 7.59 | 0.85 | 0.56 | |
| E2 | 0.05 | 0.05 | 0.13 | 0.08 | 0.32 | |
| P4 | 5.05 | 2.78 | 6.52 | 1.66 | 0.39 | |
| T | 0.33 | 0.33 | 1.33 | 0.96 | 0.14 | |
| DHT | 0.07 | 0.02 | 0.01 | 0.01 | 0.09 | |
| DOC - 11-Deoxycorticosterone, INN - 11-Deoxycortisol, CORT – Corticosterone, 17OHP – 17-Hydroxyprogesterone, A4- Androstenedione, AN – Androsterone, DHEAS – dehydroepiandrosterone sulfate, E2 - Estradiol, P4 – Progesterone, T – Testosterone, DHT – Dihydrotestosterone | ||||||
Table 1: Steroid and steroid metabolites measured in ovarian cortex culture medium from one well for each animal pooled over 4 days of culture. Data presented with mean ± SEM. Blue indicates P < 0.1 and has a tendency to be different.
| Cytokines | Control | Stair-Step | P-Value | |||
| n | 4 | 4 | ||||
| pg/mL | Mean | SEM ± | Mean | SEM ± | ||
| ANG1 | 27.29 | 11.71 | 63.66 | 25.06 | 0.39 | |
| CD40L | 4527.01 | 2986.34 | 3537.59 | 3537.59 | 0.74 | |
| DCN | 1307.68 | 320.87 | 996.54 | 282.96 | 0.77 | |
| INFβ | 0.36 | 0.36 | 0.002 | 0.002 | 0.85 | |
| IL18 | 0.00 | 0.00 | 1832.89 | 1265.33 | 0.13 | |
| LIF | 97.45 | 88.12 | 337.58 | 231.25 | 0.54 | |
| RANTES | 698.06 | 322.59 | 1254.13 | 811.19 | 0.56 | |
| INFγ | 4.49 | 1.37 | 3.68 | 0.65 | 0.77 | |
| IL13 | 501.21 | 285.07 | 810.81 | 159.67 | 0.25 | |
| IL21 | 24.38 | 9.51 | 18.52 | 6.17 | 0.56 | |
| IL1F5 | 5.07 | 3.85 | 5.40 | 1.70 | 0.56 | |
| TNFα | 61.45 | 35.00 | 44.91 | 13.41 | 0.77 | |
| ANG1 – Angiopoietin 1, CD40L – CD40 Ligand, DCN – Decorin, IFNβ – Interferon Beta 1, IL18 – Interleukin-18, LIF – Leukemia inhibitory factory, RANTES – Regulated on Activation, Normal T Cell Expressed and Secreted, IFNγ – Interferon Gamma, IL13 – Interleukin 13, IL21 – Interleukin 21, IL1F5 – Interleukin 1 family member 5, TNFα – Tumor Necrosis Factor alpha | ||||||
Table 2: Cytokine and chemokines measured in ovarian cortex culture medium from one well for each animal pooled over 4 days of culture. Data presented with mean ± SEM. Blue indicates P < 0.1-0.14 and has a tendency to be different.
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Discussion
The benefit of in vitro ovarian cortex culture, as described in this manuscript, is that the follicles develop in a normalized environment with adjacent stroma surrounding the follicles. The somatic cells and oocyte remain intact, and there is appropriate cell-to-cell communication as an in vivo model. Our laboratory has found that a 7-day culture system provides representative folliculogenesis and steroidogenesis data for the treatment of the ovarian cortex. Other ovarian tissue culture protocols have either relatively short culture periods 1-6 days7,32 or long culture periods of 10-15 days5,6,10. However, we have observed that culturing for greater than 7 days leads to tissue degradation, reduced steroidogenesis, and potentially an increased likelihood of contamination (data not shown). Culturing ovarian cortex following ovariectomy also provides direct insight into the ovarian microenvironment within that particular animal. This is of interest to our research laboratory as we have identified changes in follicular development after nutritional regimes were imposed after weaning and leading to puberty in heifers16.
Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors from somatic cells and cumulus cell-oocyte communication33. Tissue culture, such as ovarian cortex culture, offers a controlled environment to investigate the mechanistic role of these factors and the endocrine milieu in the ovarian microenvironment. Ovaries can be collected from animals that have gone through an in vivo treatment or are genetically altered to determine effects on follicle progression.
Ovaries can also be collected from a local abattoir (1 h away) and transported at 37 °C in a thermos containing PBS with antibiotic. If transport is longer (e.g., overnight), the ovaries are shipped or transported on ice22. Similar results have been observed whether the transport of ovaries happens on ice overnight or at 37 °C with short-term transport in a thermos. The 37 °C with a thermos transport allows for harvest of oocytes from this tissue for in vitro maturation (IVM) or in vitro fertilization (IVF)34. Other studies have found that transporting tissue at temperatures between 2-8 °C have also been used for fertility preservation in reproductive tissues35,36,37. Yet other studies have used ovaries transported at 34-37 °C and on ice and have not observed differences in bovine tissue culture38.
If ovarian cortex culture tissue does not look healthy after culture, this could be due to the pieces of ovarian cortex being too large. A critical step in the protocol is to ensure that the ovarian cortex pieces are no larger than 0.5-1 mm3. We utilize a ruler to measure the pieces and use a scale within the dissecting microscope to determine size of the ovarian cortex pieces (Figure 2C). Others utilize specific equipment (see Table of Materials) for uniform thickness and length/width, respectively26. If these pieces of equipment are not available, then plastic squares can be used to as a template to obtain uniform thickness and ensure similar size pieces27.
To ensure cut pieces that are only from the cortex and do not have medulla after washing the ovary we place it in the 60 mm dish and cut in half. The cortex and medulla are very different histologically as seen previously in Abedal-Majed, 202016. Each half of the ovary is filleted with the blade to ensure that only cortex is removed from the ovary and the medulla remains. If individuals are just starting to perfect this cutting technique, they can also use neutral red to closely see the histology of each half of the ovary39. This will allow for development of landmarks as their cutting technique, improves. Furthermore, they can use instruments that can cut uniform thickness (see Table of Materials) as stated above26,27.
Ovarian cortex medium should be light pink. We have observed that the pH of the ovarian cortex culture medium changes quickly in some animals, thus, the majority (70%) of the ovarian cortex medium should be changed every 24 h to promote culture health. Previous papers have discussed changing half of the medium daily40. Our reason for changing a majority of the media in the current manuscript was to promote the health of the ovarian cortex cultures. Drops surrounding the ovarian cortex pieces remain and there were no negative effects of medium change on the culture. Furthermore, this allowed us to analyze more hormones, cytokines, and chemokines for each animal to generate insight into the ovarian microenvironment.
This tissue culture protocol offers several advantages. Culturing ovarian cortex allows follicles to develop in an environment that is similar to in vivo. Follicles remain supported by the surrounding stroma and communication between somatic cells and cumulus cell-oocyte continues. The usage of culture well inserts enables the ovarian cortex to rest upon the culture medium without being submerged, thereby preventing the tissue from binding to the plastic base of the well plate. Another advantage is the culture window. The 7-day culture window described in the protocol provides representative hormone and growth factor data. In addition, folliculogenesis continues to progress in this in vitro environment as we have counted fewer early-staged follicles (primordial) and more late-staged follicles (secondary, antral) after 7 days of culture16. Previous ovarian tissue culture protocols have utilized relatively short (1-6 days7,32) or long (10-15 days5,6,10) culture periods. Shorter culture windows have been used to investigate primordial follicle activation in fetal bovine ovarian cortex41. In non-human primates, a 20-day culture period was used to evaluate the ability of the primate primordial follicles to survive and initiate growth in vitro in serum-free medium18. However, we have observed increased tissue degradation when culturing ovarian bovine cortex for longer than 7 days in serum-free medium. We have also determined in analysis of daily samples that steroid concentrations are decreased after 4 days of culture (data not shown). A longer culture window can also increase the possibility of contamination in ovarian cortex cultures. Therefore, major effects can be measured by 7 days of culture15 and time of culture may be dependent on the animal model and scientific question addressed.
While several advantages of this bovine ovarian cortex protocol exist, there are a few limitations. One limitation is the quantity of ovarian cortical tissue and culture medium volume collected during ovarian cortical culture. The small size of the ovarian cortex pieces allows for a limited number of tissue sections to be used for staining purposes (H&E, immunofluorescence, etc.). To perform RT-PCR, a minimum of four cortex pieces are needed16. Furthermore, the low volume of culture medium collected may limit the amount of analyses conducted. To combat these limitations, we suggest culturing several replicates per ovary to provide an adequate supply of culture medium and tissue for histology, assays, and PCR. Quite often we culture several pieces of each ovary from one cow/heifer to obtain more tissue for further analysis and to obtain increased culture medium to measure cortex secretion of hormones/cytokines/chemokines. Preantral follicles are more diffuse in older cow ovaries than younger females (heifers). Thus, a potential limitation is obtaining preantral follicles on all ovarian cortex pieces that are cultured. Several ways to mitigate this limitation is to obtain more ovarian cortex pieces and to culture additional wells for each animal or to use neutral red to visualize follicles and ensure that all cortex pieces contain early preantral follicles. A third limitation of the ovarian cortex culture is that any contamination of the culture system makes the media and cortex pieces unusable for analysis. Thus, several ways to maintain a sterile environment is to filter the medium used in the culture. Prior to processing and cutting ovarian cortex pieces make sure that the clean bench has been sterilized with 70% ethanol, and the air flow has been running for at least 30 min prior to dissections. Also, if using an absorbent pad to enable easier clean-up (Figure 1), ensure that the UV light has been turned on for 30 min prior to placing any tissue in the clean bench to sterilize the pad and clean bench. Finally, all media changes should be conducted in a biosafety cabinet with adequate airflow, sterile instruments, and changing pipet tips to ensure only sterile tips are introduced into the medium to be placed in wells for ovarian cortex culture.
Application of this technique will aid in understanding the ovarian microenvironment and may start to unravel mechanisms involved in female reproductive disorders that involve altered follicular development. For example, the etiology of polycystic ovary syndrome (PCOS) and aspects of premature ovarian failure (POF) remain unclear. Since cows are mono-ovulatory, they make an excellent model to understand factors that affect follicle progression and arrest in other mono-ovulatory species (e.g., humans and non-human primates). Furthermore, this ovarian cortex culture method may also prove beneficial for testing potential therapeutics that may improve follicle-mediated disorders resulting in infertility in women.
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Disclosures
The authors have nothing to disclose.
Acknowledgments
This research was supported by National Institute of Food and Agriculture 2013-67015-20965 to ASC, University of Nebraska Food for Health Competitive Grants to ASC. United States Department of Agriculture Hatch grant NEB26-202/W3112 Accession #1011127 to ASC, Hatch–NEB ANHL Accession #1002234 to ASC. Quantitative Life Sciences Initiative Summer Postdoctoral Scholar Support – COVID-19 Award for summer funding for CMS.
The authors would like to extend their appreciation to Dr. Robert Cushman, U.S. Meat Animal Research Center, Clay Center, NE to thank him for providing the ovaries in a previous publication, which were then used in the current paper as a proof of concept in validating this technique.
Materials
| Name | Company | Catalog Number | Comments |
| #11 Scapel Blade | Swann-Morton | 303 | Scaple Blade |
| #21 Scapel Blade | Swann-Morton | 307 | Scaple Blade |
| 500mL Bottle Top Filter | Corning | 430514 | Bottle Top Filter 0.22 µm pore for filtering medium |
| AbsoluteIDQ Sterol17 Assay | Biocrates | Sterol17 Kit | Samples are sent off to Biocrates and steroid panels are run and results are returned |
| Androstenedione Double Antibody RIA Kit | MPBio | 7109202 | RIA to determine androstenedione from culture medium |
| Belgium A4 Assay Kit | DIA Source | KIP0451 | RIA to determine androstenedione from culture medium |
| Bovine Cytokine Array Q3 | RayBiotech | QAB-CYT-3-1 | Cytokine kit to determine cytokines from culture medium |
| cellSens Software Standard 1.3 | Olympus | 7790 | Imaging Software |
| Insulin-Transferrin-Selenium-X | Gibco ThermoFisher Scientific | 5150056 | Addative to the culture medium |
| Leibovitz's L-15 Medium | Gibco ThermoFisher Scientific | 4130039 | Used for tissue washing on clean bench, and in the biosafety cabniet |
| Microscope | Olympus | SZX16 | Disection microscope used for imaging tissue culture pieces |
| Microscope Camera | Olympus | DP71 | Microscope cameraused for imaging tissue culture pieces |
| Millicell Cell Culture Inserts 0.4µm, 12,mm Diameter | Millipore Sigma | PICM01250 | Inserts that allow the tissue to rest against the medium without being submerged in it |
| Multiwell 24 well plate | Falcon | 353047 | Plate used to hold meduim, inserts, and tissues |
| Petri dish 60 x 15 mm | Falcon | 351007 | Petri dish used for washing steps prior to culture |
| Phosphate-Buffered Saline (PBS 1X) | Corning | 21-040-CV | Used for tissue washing |
| SAS Version 9.3 | SAS Institute | 9.3 TS1M2 | Statistical analysis software |
| Thomas Stadie-Riggs Tissue Slicer | Thomas Scientific | 6727C10 | Tissue slicer for preperation of thin uniform sections of fresh tissue |
| Waymouth MB 752/1 Medium | Sigma-Aldrich | W1625 | Medium used for tissue cultures |
References
- Braw-Tal, R., Yossefi, S. Studies in vivo and in vitro on the initiation of follicle growth in the bovine ovary. Journal of Reproduction and Fertility. 109, 165-171 (1997).
- Nilsson anEdson, M. A., Nagaraja, A. K., Matzuk, M. M. The mammalian ovary from genesis to revelation. Endocrine Reviews. 30 (6), 624-712 (2009).
- Fortune, J. E., Cushman, R. A., Wahl, C. M., Kito, S. The primordial to primary follicle transition. Molecular and Cellular Endocrinology. 163, 53-60 (2000).
- Ireland, J. J. Control of follicular growth and development. Journal of Reproduction and Fertility. 34, 39-54 (1987).
- Higuchi, C. M., Maeda, Y., Horiuchi, T., Yamazaki, Y. A simplified method for three-dimensional (3-D) ovarian tissue culture yielding oocytes competent to produce full-term offspring in mice. PLoS One. 10 (11), e0143114 (2015).
- Ramezani, M., Salehnia, M., Jafarabadi, M. Short term culture of vitrified human ovarian cortical tissue to assess the cryopreservation outcome: molecular and morphological analysis. Journal of Reproduction & Infertility. 18 (1), 162-171 (2017).
- McLaughlin, M., Telfer, E. Oocyte development in bovine primordial follicles is promoted by activin and FSH within a two-step serum-free culture system. Reproduction. 139 (6), 971-978 (2010).
- Stefansdottir, A., Fowler, P. A., Powles-Glover, N., Anderson, R. A., Spears, N. Use of ovary culture techniques in reproductive toxicology. Reproductive Toxicology. 49, 117-135 (2014).
- Bromfield, J. J., Sheldon, I. M. Lipopolysaccharide reduces the primordial follicle pool in the bovine ovarian cortex ex vivo and in the murine ovary in vivo. Biology of Reproduction. 88 (4), 1-9 (2013).
- Franks, S., Stark, J., Hardy, K. Follicle dynamics and anovulation in polycystic ovary syndrome. Humane Reproduction Update. 14 (4), 367-378 (2008).
- Desmeules, P., Devine, P. J. Characterizing the ovotoxicity of cyclophosphamide metabolites on cultured mouse ovaries. Toxicological Sciences. 90 (2), 500-509 (2006).
- Bott, R. C., McFee, R. M., Clopton, D. T., Toombs, C., Cupp, A. S. Vascular endothelial growth factor and kinase domain region receptor are involved in both seminiferous cord formation and vascular development during testis morphogenesis in the rat. Biology of Reproduction. 75, 56-67 (2006).
- Baltes-Breitwisch, M. M., et al. Neutralization of vascular endothelial growth factor antiangiogenic isoforms or administration of proangiogenic isoforms stimulates vascular development in the rat testis. Reproduction. 140 (2), 319-329 (2010).
- McFee, R. M., et al. Inhibition of vascular endothelial growth factor receptor signal transduction blocks follicle progression but does not necessarily disrupt vascular development in perinatal rat ovaries. Biology of Reproduction. 81, 966-977 (2009).
- Artac, R. A., et al. Neutralization of vascular endothelial growth factor antiangiogenic isoforms is more effective than treatment with proangiogenic isoforms in stimulating vascular development and follicle progression in the perinatal rat ovary. Biology of Reproduction. 81, 978-988 (2009).
- Abedal-Majed, M. A., et al. Vascular endothelial growth factor A isoforms modulate follicle development in peripbertal heifers independent of diet through diverse signal transduction pathways. Biology of Reproduction. 102 (3), 680-692 (2020).
- Wandji, S. A., Srsen, V., Voss, A. K., Eppig, J. J., Fortune, J. E. Initiation in vitro of bovine primordial follicles. Biology of Reproduction. 55, 942-948 (1996).
- Wandji, S. A., Srsen, V., Nathanielsz, P. W., Eppig, J. J., Fortune, J. E. Initiation of growth of baboon primordial follicles in vitro. Human Reproduction. 12 (9), 1993-2001 (1993).
- Yang, M. Y., Fortune, J. E. Testosterone stimulates the primary to secondary follicle transition in bovine follicles in vitro. Biology of Reproduction. 75, 924-932 (2006).
- Fortune, J. E., Kito, S., Wandji, S. A., Srsen, V. Activation of bovine and baboon primordial follicles in vitro. Theriogenology. 49, 441-449 (1998).
- Yang, M. Y., Fortune, J. E. Vascular endothelial growth factor stimulates the primary to secondary follicle transition in bovine follicles in vitro. Molecular Reproduction and Development. 74, 1095-1104 (2007).
- Barberino, R. S., Silva, J. R. V., Figueiredo, J. R., Matos, M. H. T. Transport of domestic and wild animal ovaries: a review of the effects of medium, temperature, and periods of storage on follicular viability. Biopreservation and Biobanking. 17 (1), 84-90 (2019).
- Summers, A. F., et al. Altered theca and cumulus oocyte complex gene expression, follicular arrest and reduced fertility in cows with dominant follicle follicular fluid androgen excess. PLoS One. 9 (10), e110683 (2014).
- Koal, T., Schmiederer, D., Pham-Tuan, H., Rohring, C., Rauh, M. Standardized LC-MS/MS based steroid hormone profile analysis. The Journal of Steroid Biochemistry and Molecular Biology. 129, 129-138 (2012).
- Poole, R. K., Brown, A. R., Pore, M. H., Pickworth, C. L., Poole, D. H. Effects of endophyte-infected tall fescue seed and protein supplementation on stocker steers: II. Adaptive and innate immune function. Journal of Animal Science. 97 (10), 4160-4170 (2019).
- Laronda, M., et al. Alginate encapsulation supports the growth and differentiation of human primordial follicles within ovarian cortical tissue. Journal of Assisted Reproduction and Genetics. 31 (8), 1013-1028 (2014).
- Silber, S. J., et al. A series of monozygotic twins discordant for ovarian failure: ovary transplantation (cortical versus microvascular) and cryopreservation. Human Reproduction. 23 (7), 1531-1537 (2008).
- Wiedemann, C., Zahmel, J., Jewgenow, K. Short-term culture of ovarian cortex pieces to assess the cryopreservation outcome in wild fields for genome conservation. BMC Veterinary Research. 9 (37), (2013).
- Baufeld, A., Vanselow, J. Increasing cell plating density mimics an early post-LH stage in cultured bovine granulosa cells. Cell and Tissue Research. 354 (3), 869-880 (2013).
- Shimizu, T., Miyamoto, A. Progesterone induces the expression of vascular endothelial growth factor (VEGF) 120 and Flk-1, its receptor, in bovine granulosa cells. Animal Reproduction Science. 102 (3-4), 228-237 (2007).
- Tepekoy, F., Akkoyunlu, G. The effect of FSH and activin A on Akt and MAPK1/3 phosphorylation in cultured bovine ovarian cortical strips. Journal of Ovarian Research. 9 (13), 1-9 (2016).
- Beck, K., Singh, J., Arshud Dar, M., Anzar, M. Short-term culture of adult bovine ovarian tissues: chorioallantoic membrane (CAM) vs. traditional in vitro culture systems. Reproductive Biology and Endocrinology. 16 (1), 21 (2018).
- Eppig, J. J. Oocyte control of ovarian follicular development and function in mammals. Reproduction. 122 (6), 829-838 (2001).
- Paczkowski, M., Silva, E., Schoolcraft, W. B., Krisher, R. L. Comparative importance of fatty acid beta-oxidation to nuclear maturation, gene expression, and glucose metabolism in mouse, bovine, and porcine cumulus oocyte complexes. Biology of Reproduction. 88 (5), 1-11 (2013).
- Raffel, N., et al. Is ovarian tissue transport at supra-zero temperatures compared to body temperature optimal for follicle survival? In Vivo. 34 (2), 533-541 (2020).
- Duncan, F., et al. Ovarian tissue transport to expand access to fertility preservation: from animals to clinical practice. Reproduction (Cambridge, England). 152 (6), R201-R210 (2016).
- Liebenthron, J., et al. Overnight ovarian tissue transportation for centralized cryobanking: a feasible option. Reproductive BioMedicine Online. 38 (5), 740-749 (2019).
- Mohammed, B. T., Donadeu, F. X.
- Langbeen, A., et al. Effects of neutral red assisted viability assessment on the cryotolerance of isolated bovine preantral follicles. Journal of Assisted Reproduction Genetics. 31, 1727-1736 (2014).
- Higuchi, C. M., Maeda, Y., Horiuchi, T., Yamazaki, Y. A simplified method for three-dimensional (3-D) ovarian tissue culture yielding oocytes competent to produce full-term offspring in mice. PLoS One. 10 (11), e0143114 (2015).
- Yang, M. Y., Fortune, J. E. Changes in the transcriptome of bovine ovarian cortex during follicle activation in vitro. Physiological Genomics. 47, 600-611 (2015).
