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&#8211;NEB ANHL Accession #1002234 to ASC. Quantitative Life Sciences Initiative Summer Postdoctoral Scholar Support &#8211; 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.

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
<ol>
	<li>Waymouth MB 752/1 medium
	<ol>
		<li>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.</li>
		<li>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 &#181;m pore 33.2 cm2 500 mL bottle top filter.</li>
		<li>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.</li>
		<li>Wrap the conical tubes and stock bottle of the medium in aluminum foil and store at 4 &#176;C. This medium is light sensitive. 
		NOTE: Waymouth medium can be stored for up to 1 month.</li>
	</ol>
	</li>
	<li>Leibovitz&#39;s L-15 (LB-15) medium 
	NOTE: LB-15 medium is used to clean tissue in preparation for culture.
	<ol>
		<li>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.</li>
		<li>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 &#181;m 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 &#176;C. 
		NOTE: LB-15 medium can be stored for up to 1 month.</li>
	</ol>
	</li>
	<li>Phosphate Buffered Saline (PBS)
	<ol>
		<li>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.</li>
		<li>Make 1 L PBS with 0.1% antibiotic (see Table of Materials) while in a biological safety cabinet.</li>
	</ol>
	</li>
</ol>
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 &#176;C23 to University of Nebraska-Lincoln Reproduction Laboratory UNL (1.5 h away). (For comments on temperature of ovaries during transport please see Discussion)
<ol>
	<li>Prepare the ovarian tissue on a clean bench (Figure 1).
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Fill each Petri dish with approximately 10 mL of appropriate fluids, either PBS or LB-15.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61668/61668fig01.jpg" /> 
Figure 1:&#160;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. <a href="https://www.jove.com/files/ftp_upload/61668/61668fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Remove the prepared Waymouth and LB-15 medium from the refrigerator and warm to room temperature.</li>
	<li>Autoclave all tools to ensure sterilization prior to use.</li>
	<li>Maintain ovaries at 37 &#176;C until ovarian cortex is ready to be collected.</li>
	<li>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.</li>
	<li>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&#8211;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&#8211;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&#8211;2 mm depth, then the medulla should not be obtained. Distinct histology allows for landmarks between the cortex and the medulla.</li>
	<li>Cut the ovarian cortex strips in the third PBS wash into small, square pieces (~0.5&#8211;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.</li>
	<li>Wash ovarian cortical pieces through all three PBS with antibiotic-filled Petri dishes. Use a curved tip forceps to move pieces between washes.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Move all ovarian cortex intended for culture to the biological safety cabinet and wash once more in an LB-15-filled Petri dish.</li>
	<li>In a 24-well tissue culture plate, pipette 350 &#181;L of Waymouth medium per well.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Incubate the tissue at 37 &#176;C with 5% CO216. 
	NOTE: Others have used 38.8 &#176;C28. However, no difference has been observed in the integrity of the tissue nor in the ability of follicles to progress in 37 &#176;C tissue nor have others39,30. Thus, at this point any of these temperatures should be conducive to experiment success. Others have used 400 &#181;L of medium. Either amount is fine as long as one is consistent, and the &#160;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 &#181;L of sterile water to help reduce evaporation from other wells.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61668/61668fig02.jpg" /> 
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 &#181;L) to maintain proper pH.Approximately 250 &#181;L is obtained from each well each day (about 70% of the initial culture medium). <a href="https://www.jove.com/files/ftp_upload/61668/61668fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Media collection 
<ol>
	<li>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 &#176;C prior to medium change. Approximately 250 &#181;L is obtained from each well each day (about 70% of initial culture medium).</li>
	<li>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 &#181;L /day). Set the insert back in well and add 350 &#181;L 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.</li>
	<li>Store the collected medium from tissue culture at -20 &#176;C.</li>
</ol>
4. Imaging and downstream processing
<ol>
	<li>After 7 days of culture at 37 &#176;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.</li>
	<li>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 &#176;C.</li>
	<li>Let the ovarian cortex pieces remain immersed in Bouins (picric acid 750 mL, glacial acetic acid 50 mL, and 37%&#8211;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.</li>
</ol>

The authors have nothing to disclose.

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 ...

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&#176;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 &#181;L/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 &#34;normal&#34; or &#34;abnormal&#34; folliculogenesis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#11 Scapel Blade</td>
			<td>Swann-Morton&#160;</td>
			<td>303</td>
			<td>Scaple Blade</td>
		</tr>
		<tr>
			<td>#21 Scapel Blade</td>
			<td>Swann-Morton&#160;</td>
			<td>307</td>
			<td>Scaple Blade</td>
		</tr>
		<tr>
			<td>500mL Bottle Top Filter</td>
			<td>Corning</td>
			<td>430514</td>
			<td>Bottle Top Filter 0.22 &#181;m pore for filtering medium</td>
		</tr>
		<tr>
			<td>AbsoluteIDQ Sterol17 Assay</td>
			<td>Biocrates</td>
			<td>Sterol17 Kit</td>
			<td>Samples are sent off to Biocrates and steroid panels are run and results are returned&#160;</td>
		</tr>
		<tr>
			<td>Androstenedione Double Antibody RIA Kit</td>
			<td>MPBio</td>
			<td>7109202</td>
			<td>RIA to determine androstenedione from culture medium</td>
		</tr>
		<tr>
			<td>Belgium A4 Assay Kit&#160;</td>
			<td>DIA Source&#160;</td>
			<td>KIP0451</td>
			<td>RIA to determine androstenedione from culture medium</td>
		</tr>
		<tr>
			<td>Bovine Cytokine Array Q3</td>
			<td>RayBiotech</td>
			<td>QAB-CYT-3-1</td>
			<td>Cytokine kit to determine cytokines from culture medium</td>
		</tr>
		<tr>
			<td>cellSens Software Standard 1.3</td>
			<td>Olympus</td>
			<td>7790</td>
			<td>Imaging Software</td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium-X</td>
			<td>Gibco ThermoFisher Scientific</td>
			<td>5150056</td>
			<td>Addative to the culture medium</td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 Medium</td>
			<td>Gibco ThermoFisher Scientific</td>
			<td>4130039</td>
			<td>Used for tissue washing on clean bench, and in the biosafety cabniet&#160;</td>
		</tr>
		<tr>
			<td>Microscope&#160;</td>
			<td>Olympus</td>
			<td>SZX16</td>
			<td>Disection microscope used for imaging tissue culture pieces&#160;</td>
		</tr>
		<tr>
			<td>Microscope Camera&#160;</td>
			<td>Olympus</td>
			<td>DP71</td>
			<td>Microscope cameraused for imaging tissue culture pieces&#160;</td>
		</tr>
		<tr>
			<td>Millicell Cell Culture Inserts 0.4&#181;m, 12,mm Diameter</td>
			<td>Millipore Sigma</td>
			<td>PICM01250</td>
			<td>Inserts that allow the tissue to rest against the medium without being submerged in it</td>
		</tr>
		<tr>
			<td>Multiwell 24 well plate</td>
			<td>Falcon</td>
			<td>353047</td>
			<td>Plate used to hold meduim, inserts, and tissues</td>
		</tr>
		<tr>
			<td>Petri dish 60 x 15 mm</td>
			<td>Falcon</td>
			<td>351007</td>
			<td>Petri dish used for washing steps prior to culture</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS 1X)</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td>Used for tissue washing</td>
		</tr>
		<tr>
			<td>SAS Version 9.3</td>
			<td>SAS Institute&#160;</td>
			<td>9.3 TS1M2</td>
			<td>Statistical analysis software&#160;</td>
		</tr>
		<tr>
			<td>Thomas Stadie-Riggs Tissue Slicer</td>
			<td>Thomas Scientific</td>
			<td>6727C10</td>
			<td>Tissue slicer for preperation of thin uniform sections of fresh tissue</td>
		</tr>
		<tr>
			<td>Waymouth MB 752/1 Medium</td>
			<td>Sigma-Aldrich</td>
			<td>W1625</td>
			<td>Medium used for tissue cultures</td>
		</tr>
	</tbody>
</table>

bovine ovarian cortex tissue culture

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&#39;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.

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&#38;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 pr...

Watch this Scientific Journal Video about Bovine Ovarian Cortex Tissue Culture at JoVE.com

Bovine Ovarian Cortex Tissue Culture

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.

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors ...

Research

JoVE Journal

Biology

4.4K Views.  University of Nebraska-Lincoln. 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.

Video: Bovine Ovarian Cortex Tissue Culture

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address the requirements (e.g., mechanics and vascularity) of more intricate organs and tissues. Electrospinning is a popular technique to create fibrous scaffolds that mimic the architecture and size scale of the native extracellular matrix. These fibrous scaffolds are also useful as cell culture substrates since the fibers can be used to direct cellular behavior, including stem cell differentiation (see extensive reviews by Mauck et al. and Sill et al. for more information). In this article, we describe the general process of electrospinning polymers and as an example, electrospin a reactive hyaluronic acid capable of crosslinking with light exposure (see Ifkovits et al. for a review on photocrosslinkable materials). We also introduce further processing capabilities such as photopatterning and multi-polymer scaffold formation. Photopatterning can be used to create scaffolds with channels and multi-scale porosity to increase cellular infiltration and tissue distribution. Multi-polymer scaffolds are useful to better tune the properties (mechanics and degradation) of a scaffold, including tailored porosity for cellular infiltration. Furthermore, these techniques can be extended to include a wide array of polymers and reactive macromers to create complex scaffolds that provide the cues necessary for the development of successful tissue engineered constructs.

The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address ...

A Tissue Culture Model of Estrogen-producing Primary Bovine Granulosa Cells

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca and, in particular, of the granulosa cell layer profoundly change their morphological, physiological, and molecular characteristics and form the progesterone-producing corpus luteum that is responsible for maintaining pregnancy. Cell culture models are essential tools to study the underlying regulatory mechanisms involved in the folliculo-luteal transformation. The presented protocol focuses on the isolation procedure and cryopreservation of bovine GC from small- to medium-sized follicles (&#60; 6 mm). With this technique, a nearly pure population of GC can be obtained. The cryopreservation procedure greatly facilitates time management of the cell culture work independent of a direct primary tissue (ovaries) supply. This protocol describes a serum-free cell culture model that mimics the estradiol-active status of bovine GC. Important conditions that are essential for a successful steroid-active cell culture are discussed throughout the protocol. It is demonstrated that increasing the plating density of the cells induces a specific response as indicated by an altered gene expression profile and hormone production. Furthermore, this model provides a basis for further studies on GC differentiation and other applications.

A long-term culture model of bovine granulosa cells under serum-free conditions is described. This model allows researchers to study the effects of diverse factors and conditions as different plating densities on the characteristics of estrogen-producing bovine granulosa cells.

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca ...

Bovine Mammary Gland Biopsy Techniques

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, signaling pathways, and protein translation. This article describes two techniques for biopsy of the bovine mammary gland (MG). Three healthy Holstein dairy cows were the subjects. Before biopsies, cows were milked and subsequently restrained in a cattle chute. An analgesic (flunixin meglumine, 1.1 to 2.2 mg/kg of body weight) was administered via jugular intravenous [IV] injection 15-20 min prior to biopsy. For standing sedation, xylazine hydrochloride (0.01-0.05 mg/kg of body weight) was injected via the coccygeal vessels 5-10 min before the procedure. Once adequately sedated, the biopsy site was aseptically prepared and locally anaesthetized with 6 mL of 2% lidocaine hydrochloride via subcutaneous injection. Using aseptic technique, a 2 to 3 cm vertical incision was made using a number 10 scalpel. Core and needle biopsy tools were used. The core biopsy tool was attached to a cordless drill and inserted into the MG tissue through the incision using a clock-wise drill action. The needle biopsy tool was manually inserted into the incision site. Immediately after the procedure, an assistant applied pressure on the incision site for 20 to 25 min using a sterile towel to achieve hemostasis. Stainless steel surgical staples were used to oppose the skin incision. The staples were removed 10 days post-procedure. The main advantages of core and needle biopsies is that both approaches are minimally invasive procedures that can be safely performed in healthy cows. Milk yield following the biopsy was unaffected. These procedures require a short recovery time and result in fewer risks of complications. Specific limitations may include bleeding after the biopsy and infection on the biopsy site. Applications of these techniques include tissue collection for clinical diagnosis and research purposes, such as primary cell culture.

This article presents a bovine mammary gland biopsy using core and needle biopsy tools. Harvested tissue can be used for cell culture or to assess mammary physiology and metabolism including gene expression, protein expression, protein modifications, immunohistochemistry, and metabolite concentrations.

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, ...

Reproductive Techniques for Ovarian Monitoring and Control in Amphibians

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive behaviors and the acquisition of gametes and embryos for physiological or molecular research. Amphibians contribute to one-quarter to one-third of vertebrate research, and of interest in this context is their contribution to the scientific community&#39;s knowledge of reproductive processes and embryological development. However, most of this knowledge is derived from a small number of species. In recent times, the decimation of amphibians across the globe has required increasing intervention by conservationists. The captive recovery and assurance colonies that continue to emerge in response to the extinction risk make existing research and clinical applications invaluable to the survival and reproduction of amphibians held under human care. The success of any captive population is founded on its health and reproduction and the ability to develop viable offspring that carry forward the most diverse genetic representation of their species. For researchers and veterinarians, the ability to monitor and control ovarian development and health is, therefore, imperative. The focus of this article is to highlight the different assisted reproductive techniques that can be used to monitor and, where appropriate or necessary, control ovarian function in amphibians. Ideally, any reproductive and health issues should be reduced through proper captive husbandry, but, as with any animal, issues of health and reproductive pathologies are inevitable. Non-invasive techniques include behavioral assessments, visual inspection and palpation and morphometric measurements for the calculation of body condition indices and ultrasound. Invasive techniques include hormonal injections, blood sampling, and surgery. Ovarian control can be exercised in a number of ways depending on the application required and species of interest.

The study of amphibian biology provides valuable information on the reproductive, physiological, embryological and developmental processes that drive organisms of many taxonomic groups. Here, we present a comprehensive guide on different methodologies that can be used to study ovarian control and monitoring in amphibians.

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive ...

An Ex Vivo Tissue Culture Model of Cartilage Remodeling in Bovine Knee Explants

Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique tissue important for proper function of the synovial joint and is constituted by a dense extracellular matrix (ECM), rich in proteoglycan and type II collagen. Chondrocytes are the only cell type present within cartilage and are widespread and relatively low in number. Altered external stimuli and cellular signalling can lead to changes in ECM composition and deterioration, which are important pathological hallmarks in diseases such as osteoarthritis (OA) and rheumatoid arthritis.
Ex vivo cartilage models allow 1) profiling of chondrocyte mediated alterations of cartilage tissue turnover, 2) visualizing the cartilage ECM composition, and 3) chondrocyte rearrangement directly in the tissue. Profiling these alterations in response to stimuli or treatments are of high importance in various aspects of cartilage biology, and complement in vitro experiments in isolated chondrocytes, or more complex models in live animals where experimental conditions are more difficult to control.
Cartilage explants present a translational and easily accessible method for assessing tissue remodeling in the cartilage ECM in controllable settings. Here, we describe a protocol for isolating and culturing live bovine cartilage explants. The method uses tissue from the bovine knee, which is easily accessible from the local butchery. Both explants and conditioned culture medium can be analyzed to investigate tissue turnover, ECM composition, and chondrocyte function, thus profiling ECM modulation.

Here, we present a protocol describing isolation and culturing of cartilage explants from bovine knees. This method provides an easy and accessible tool to describe tissue changes in response to biological stimuli or novel therapeutics targeting the joint.

Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique ...

Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of these methods to non-model organisms. A series of protocols to optimize tissue collection from bats has been developed for a series of high-throughput sequencing approaches. Outlined here are protocols for the capture of bats, desired demographics to be collected for each bat, and optimized methods to minimize stress on a bat during tissue collection. Specifically outlined are methods for collecting and treating tissue to obtain (i) DNA for high molecular weight genomic analyses, (ii) RNA for tissue-specific transcriptomes, and (iii) proteins for proteomic-level analyses. Lastly, also outlined is a method to avoid lethal sampling by creating viable primary cell cultures from wing clips. A central motivation of these methods is to maximize the amount of potential molecular and morphological data for each bat and suggest optimal ways to preserve tissues so they retain their value as new methods develop in the future. This standardization has become particularly important as initiatives to sequence chromosome-level, error-free genomes of species across the world have emerged, in which multiple scientific parties are spearheading the sequencing of different taxonomic groups. The protocols outlined herein define the ideal tissue collection and tissue preservation methods for Bat1K, the consortium that is sequencing the genomes of every species of bat.

This is a protocol for the optimal tissue preparation for genomic, transcriptomic, and proteomic analyses of bats caught in the wild. It includes protocols for bat capture and dissection, tissue preservation, and cell culturing of bat tissue.

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of ...

Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular matrix. The ability of CSCs to participate in the development of endothelium constitutes an important characteristic that directly contributes to the general understanding of the mechanisms of tumorigenesis and tumor metastasis. The purpose of this work is to establish a reproducible methodology to investigate the tumor-initiation capability of ovarian cancer stem cells (OCSCs). Herein, we examined the neovascularization mechanism between endothelial cells and OCSCs along with the morphological changes of endothelial cells using the in vitro co-culture model NICO-1. This protocol allows visualization of the neovascularization step surrounding the OCSCs in a time course manner. The technique can provide insight regarding the angiogenetic properties of OCSCs in tumor metastasis.

Ovarian cancer stem cells (OCSC) are responsible for cancer initiation, recurrence, therapeutic resistance, and metastasis. The OCSC vascular niche is considered to promote self-renewal of OCSCs, leading to chemoresistance. This protocol provides the basis for establishing a reproducible OCSC vascular niche model in vitro.

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular ...

Unveiling Inflammatory Arthritis&#58; Exploring Functions of Synovial Cells

Isolation and Culture of Primary Synovial Macrophages and Fibroblasts from Murine Arthritis Tissue

Rheumatoid arthritis is an autoimmune disease that leads to chronic inflammation of joints. Synovial macrophages and synovial fibroblasts have central roles in the pathogenesis of rheumatoid arthritis. It is important to understand the functions of both cell populations to reveal the mechanisms underlying pathological progression and remission in inflammatory arthritis. In general, in vitro experimental conditions should mimic the in vivo environment as much as possible. Primary tissue-derived cells have been used in experiments characterizing synovial fibroblasts in arthritis. In contrast, in experiments investigating the biological functions of macrophages in inflammatory arthritis, cell lines, bone marrow-derived macrophages, and blood monocyte-derived macrophages have been used. However, it is unclear whether such macrophages actually reflect the functions of tissue-resident macrophages. To obtain resident macrophages, previous protocols were modified to isolate and expand both primary macrophages and fibroblasts from synovial tissue in an inflammatory arthritis mouse model. These primary synovial cells may be useful for in vitro analysis of inflammatory arthritis.

Author Spotlight: Isolation and Culture of Primary Synovial Macrophages and Fibroblasts from Murine Arthritis Tissue  

The present study provides a modified protocol to isolate synovial macrophages and fibroblasts from murine inflammatory arthritis tissue.

Rheumatoid arthritis is an autoimmune disease that leads to chronic inflammation of joints. Synovial macrophages and synovial fibroblasts have central ...

Live Cell Imaging of Lipid Droplet Size and Fusion Using Oil Red O and BODIPY

Evaluation of Lipid Droplet Size and Fusion in Bovine Hepatic Cells

Lipid droplets (LDs) are organelles that play an important role in lipid metabolism and neutral lipid storage in cells. They are associated with a variety of metabolic diseases, such as obesity, fatty liver disease, and diabetes. In hepatic cells, the sizes and numbers of LDs are signs of fatty liver disease. Moreover, the oxidative stress reaction, cell autophagy, and apoptosis are often accompanied by changes in the sizes and numbers of LDs. As a result, the dimensions and quantity of LDs are the basis of the current research regarding the mechanism of LD biogenesis. Here, in fatty acid-induced bovine hepatic cells, we describe how to use oil red O to stain LDs and to investigate the sizes and numbers of LDs. The size distribution of LDs is statistically analyzed. The process of small LDs fusing into large LDs is also observed by a live cell imaging system. The current work provides a way to directly observe the size change trend of LDs under different physiological conditions.

Author Spotlight: Evaluation of Lipid Droplet Size and Fusion in Bovine Hepatic Cells  

The present protocol describes how to use oil red O to dye lipid droplets (LDs), calculate the size and number of LDs in a fatty acid-induced fatty hepatocyte model, and use BODIPY 493/503 to observe the process of small LDs fusing into large LDs by live cell imaging.

Lipid droplets (LDs) are organelles that play an important role in lipid metabolism and neutral lipid storage in cells. They are associated with a variety ...