Evaluation of the Estrous Cycle, Reproductive Tract, and Mammary Gland in Female Mice

Justin D. Vidal1, Adam J. Filgo1

1 MPI Research, Mattawan, Michigan
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/cpmo.35
Online Posting Date:  December, 2017
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Evaluation of the female reproductive system is an important part of basic reproductive biology research, toxicology testing, and mutant mouse phenotype assessment. The female reproductive system is dynamic and the onset of puberty and the normal changes observed during estrous cyclicity can create challenges for an investigator. Experimental work in the female mouse requires an understanding of the potential impact of the estrous cycle and tracking normal changes throughout the cycle allows for control of this key variable. The estrous cycle can be evaluated using vaginal cytology, which provides the researcher a daily assessment of the entire reproductive endocrine axis and allows for samples to be collected at precise times within the cycle. These protocols describe the basic approach to evaluating the onset of cyclicity, tracking the normal cycle, and collection and microscopic evaluation of the reproductive system and mammary gland in the mouse. © 2017 by John Wiley & Sons, Inc.

Keywords: cytology; estrous cycle; mammary gland; mouse; ovary; uterus; vagina

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Table of Contents

  • Introduction
  • Basic Protocol 1: Evaluation of the Mouse Estrous Cycle and Reproductive Tract
  • Basic Protocol 2: Mouse Mammary Gland Collection and Processing (Whole Mount and Routine Microscopy)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Evaluation of the Mouse Estrous Cycle and Reproductive Tract

  • Female mice (21 days of age through sexual maturity)
  • 1× phosphate‐buffered saline (PBS, Fisher Scientific)
  • Modified Wrights‐Giemsa stain
  • Permount
  • 10% neutral buffered formalin (NBF)
  • 70% ethanol
  • Xylene
  • Paraffin
  • Hematoxylin and eosin
  • Microscopic slides and coverslips
  • Wax pencil or hydrophobic pen
  • Disposable, fine‐tip transfer pipettes
  • Light microscope
  • Slide warmer
  • Automatic stainer, optional
  • Necropsy instruments (scalpel, scissors, forceps, etc.)
  • Tissue capsules/cassettes
  • Containers for tissue fixation
  • Manual or automated rotary microtome

Basic Protocol 2: Mouse Mammary Gland Collection and Processing (Whole Mount and Routine Microscopy)

  • Euthanized mouse
  • 70%, 95%, and 100% ethanol
  • 10% neutral buffered formalin (NBF)
  • Carnoy's fixative (see recipe)
  • Carmine Alum stain (see recipe)
  • Xylene
  • Permount
  • Paraffin
  • Hematoxylin and eosin
  • Charged slides
  • Dissection board
  • T pins
  • Dissection instruments, e.g., standard dissecting forceps, fine curved forceps, fine curved scissors
  • Tissue capsules/cassettes
  • Piece of cardboard or corkboard
  • Parafilm
  • Slide trays
  • Weighted objects: 50‐ml conical tubes filled with water and capped or 400‐ to 500‐g weights
  • Coplin jars
  • Disposable pipettes
  • Cover slips
  • Cotton swab
  • Dissecting scope
  • Manual or automated rotary microtome
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Literature Cited

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  Cora, M. C., Kooistra, L., & Travlos, G. (2015). Vaginal cytology of the laboratory rat and mouse: Review and criteria for the staging of the estrous cycle using stained vaginal smears. Toxicologic Pathology, 43(6), 776–793. doi: 10.1177/0192623315570339.
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  Tucker, D. K., Foley, J. F., Hayes‐Bouknight, S. A., & Fenton, S. E. (2016). Preparation of high‐quality hematoxylin and eosin–stained sections from rodent mammary gland whole mounts for histopathologic review. Toxicologic Pathology, 44(7), 1059–1064. doi: 10.1177/0192623316660769.
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Internet Resources
  This method describes how to dissect and assess mammary gland development and function from mice.
  This protocol describes a reliable method for the quantitative assessment of mammary gland branching characteristics.
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