INTRODUCTION

Important Background Information 

What is the Corpus Luteum?

• ​The corpus luteum is a transient endocrine gland found in mammalian ovaries after the ovulation of the oocyte 
• Luteal Cells are primarily responsible for the production of the steroid hormone progesterone 
• An organelle called the mitochondrion is the site of steroid hormone synthesis, or steroidogenesis, in luteal cells
• ​Mitochondria actively maintain a highly negative potential across their inner membrane
  • A mitochondrion’s health directly correlates to its membrane potential, and maintaining it is key to cellular survival

What is Progesterone? 

• Progesterone is responsible for establishment and maintenance of pregnancy
• Progesterone steroidogenesis begins when cytosolic lipid droplets in the cell undergo facilitated diffusion into the mitochondria through the StARD1 enzyme
•  The enzyme Cyp11A1 then converts the cholesterol into a steroid intermediate called pregnenolone
•  Pregnenolone then exits the mitochondria and travels to the endoplasmic reticulum, where the enzyme 3B-HSD converts it to progesterone

What is Prostaglandin? 

• If a female cow does not become pregnant, or does not detect a viable embryo (maternal recognition), the hormone prostaglandin is produced​
• Prostaglandin acts through a signal transduction pathway to lower the rate of transcription of the 3B-HSD enzyme, ultimately resulting in a decrease in progesterone production
  
• The corpus luteum also becomes smaller
  
  • ​One of the ways that prostaglandin induces structural and functional regression of the corpus luteum is through decreasing luteal blood flow, resulting in hypoxia
    

WHY DOES THIS MATTER?

Cows serve as an important source of food, primarily dairy and beef, making the bovine industry one of the United States’ most vital enterprises. In cows, the rate of maternal recognition is very low--only about 40% in dairy cows and 65% in beef cattle. Cow miscarriages are time-consuming and expensive for beef and dairy farmers: The economic loss resulting from each pregnancy loss was estimated at approximately $2,333. To make the industry more cost-effective, researchers are investigating ways to improve the rate of maternal recognition.

Previous studies have shown that supplementing the diet of female cows with omega-3 polyunsaturated fatty acids, administered in the form of fish oil, decreases the sensitivity of bovine luteal cells to prostaglandin by inducing changes in the lipid structures. The purpose of our study is to evaluate the cellular effects of these fish oil supplementation treatments on the mitochondria of bovine luteal cells in hypoxic conditions.

EXPERIMENTAL SETUP 

Using one hypoxia chamber, one set of luteal cells will be exposed to 5% oxygen. Another set will be exposed to 20% oxygen in an incubator. In each oxygenated chamber, there will be a sample of cells treated with vegetable oil, a sample treated with bovine serum albumin (BSA), and another sample treated with fish oil. Vegetable oil will be used because it is polyunsaturated like fish oil.

​The independent variable in this study is treatment (fish oil, vegetable oil, or control medium) while the dependent variable is mitochondrial membrane potential. 

THE HYPOTHESES 

HYPOTHESIS

• When grown under hypoxic conditions, bovine luteal cells will demonstrate greater mitochondrial membrane potential when treated with fish oil as opposed to cells treated with vegetable oil or BSA control medium

NULL HYPOTHESIS

• When grown under hypoxic conditions, bovine luteal cells will not demonstrate greater mitochondrial membrane potential when treated with fish oil as opposed to BSA control medium or vegetable oil 
  

PREDICTIONS

• We predict that the cell samples in the 20% oxygen environment will both be healthy--that is, that the mitochondrial membrane potential will be the same
• However, we predict that in the 5% oxygen chamber, the sample treated with fish oil will have a higher mitochondrial membrane potential than the cells treated with vegetable oil or BSA

QUANTIFYING RESULTS

How is Mitochondrial Membrane Potential Measured?

 Mitochondrial membrane potential can be measured by both flow cytometry and confocal microscopy. In a confocal microscope, the final image has the same focus as or the focus corresponds to the point of focus in the object. This means that we can more precisely show colocalizations of the signals from our cell samples to obtain clearer images. While microscopy can be used to generate highly specific images of samples, flow cytometry sorts cells in a sample according to size, complexity, and fluorescence to generate histogram plots with greater amounts of data.