Research Methods for Studying Mammary Gland Biology

Microscopic methods include…

• Whole mounts

• Histology

• Morphometry

Describe Whole Mounts

• Fix and stain entire gland for viewing the complete unsectioned organ

• Study developing tissues and organs in the embryo and evaluate sub-gross changes in fully developed tissues

• Method includes: mammary gland removal → fixation on slide → staining → clearing and mounting → observe under a microscope

Describe Histology

• Fix and stain small pieces of tissues

• Good for observing fine structures (i.e., alveoli) and cells

• Qualitative

• Method includes: mammary gland removal → fixation of small pieces on slide → staining → clearing and mounting → observe under a microscope

Describe Morphometry

• A variation of histology giving quantitative data

• Involves the examination of numerous sections of mammary gland to obtain:

  • 1. the cell numbers of a particular cell type

  • 2. volume occupied by a particular cell or structure (i.e., parenchyma vs. stroma)

• Very tedious

Biochemical methods include…

• Composition analysis

• DNA

• Hydroxyproline

• ³H Thymidine incorporation

These methods can be applied to the whole gland or to its dissected parts

Composition Analysis

determination of fat and protein content

DNA as a biochemical method

• DNA content of a cell is constant

• DNA content give an index of mammary growth

Hydroxyproline

hydroxylation of proline is a post-translational modification of proteins and occurs only in connective tissue → index of connective tissue

³H Thymidine Incorporation

• Culture mammary tissue pieces with ³H Thymidine

• Thymidine is a precursor for DNA synthesis

• A dynamic measure of cellular proliferation

• When combined with autoradiography and histology → identification of types of cells undergoing proliferation within the gland

Imaging Methods

• Magnetic Resonance Imaging (MRI)

• X-ray Computed Tomography (X-ray CT)

Describe Imaging Methods

• Provide real images of the mammary

• Yield estimates of the total volume of the mammary as well as estimates of the true volume of parenchymal tissue

Describe In Vitro Models

• Experimentation performed not in a living organism, but in a controlled environment

• e.g. in a tube or a petri dish

• Study effects of an experimental variable on specific cells or tissues of an organism

• Better suited for deducing biological mechanisms of action, so systemic effect

• Low cost

• Testing conditions doesn’t equal the conditions inside an organism

• Results may be “artificial” and misleading

• Some products are secreted in medium (i.e. caseins), others are not (lipids)

• Rates of synthesis in vitro are usually only a fraction of in vivo rates

• In vitro studies are usually followed by in vivo studies

What are in vitro model systems?

• Whole organ culture of the mammary gland

• Tissue explant culture

• Cell culture

Describe Whole Organ Culture of the Mammary Gland

Whole gland is harvested and placed in a cell culture dish containing growth media or treatment media

Describe Tissue Explant Culture

Tissue is harvested at slaughter, by surgery and biopsy → sliced → very small pieces (1mm³) are placed on filter papers floating on culture media containing substrates and hormones

Describe Cell Culture

• Grow cells under controlled conditions in petri dishes or flasks

• Cells can be either cell lines (grown in culture for many generations) or primary cells

• Cell can be attached on the bottom surface (2D) or on or embedded into extracellular matrix components (e.g. collagen) (3D) to improve their ability to synthesize milk components

• Used extensively to study effects of hormones, substrates and extracellular matrix on metabolic activity of cells

Describe Primary Cell Culture

the culture of cells isolated from tissue taken directly form the living organism (e.g. biopsy material)

Describe In Vivo Model Systems

• “Experimentation using a whole, living organism as opposed to a partial or dead organism”

• Include animal testing and clinical trails

• Observe the overall effects of an experiment on a living subject. Results are more “real”

Other In Vivo Model Systems

• Hormone treatment of normal animals

• Removal of an endocrine gland +/- replacement therapy

Transgenic model

• Overproduction of a gene product

• By introducing one or more copies of a gene of interest into the genome of an organism

Gene Knockout

• Total removal of a gene product

• By eliminating both copies of a gene in an organism (mouse)

What are the techniques to study milk synthesis?

• Mammary uptake in vivo

• Metabolic tracers

Mammary uptake in vivo

• measure metabolite concentration in artery and vein serving the mammary gland and determine blood flow

• Uptake= {[metabolite]_AR-[metabolite]_vein}x blood flow

Metabolic Tracers

• Can be radioactive isotope (e.g., [ring-2,6-³H] phenylalanine) or stable isotope (e.g., [1-¹³C] phenylalanine)

• Combines with other techniques

• Determine precursor → product relationship and rate of production/utilization of various metabolites