Bovine Ovarian Cortex Tissue Culture

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 inputs^1,2,3,4. 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 fo....

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 o.......

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 .......

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