Name: bovine ovarian cortex tissue culture
Uploaded: 2021-01-14T14:00:00
Description: Watch this Scientific Journal Video about Bovine Ovarian Cortex Tissue Culture at JoVE.com

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 o...

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.

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	<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 ...

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