Autoimmune Ovarian Disease Induced by Immunization with Zona Pellucida (ZP3) Peptide

Kenneth Tung1, Sally Agersborg1, Harini Bagavant1, Kristine Garza1, Kevin Wei1

1 University of Virginia, Charlottesville, Virginia
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 15.17
DOI:  10.1002/0471142735.im1517s49
Online Posting Date:  August, 2002
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Abstract

Autoimmune ovarian disease is a known cause of human premature ovarian failure. An experimental murine model can be created by immunization with a peptide of ZP3, an ovary‐specific glycoprotein. Mice injected with the ZP3 peptide develop histological evidence of ovarian inflammation (oophoritis), as well as antibody to the zona pellucida and T cell responses to the peptide. This unit describes the immunologic properties of the ZP3 peptides, the method of active autoimmune oophoritis induction, and the evaluation and semiquantitation of ovarian pathology. Surgical manipulations of the ovaries have been instrumental in the novel application of this autoimmune model; thus the most useful operations are also detailed. Surgical procedures are included for adult oophrectomy, neonatal ovariectomy, and implantation of ovarian grafts into mice without endogenous ovaries.

     
 
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Table of Contents

  • Basic Protocol 1: Active Induction of Autoimmune Ovarian Disease with a ZP3 Peptide
  • Support Protocol 1: Evaluation of Autoimmune Ovarian Disease
  • Support Protocol 2: Adult Oophorectomy in Mice
  • Support Protocol 3: Neonatal Ovariectomy in Mice
  • Support Protocol 4: Implantation of Ovarian Grafts in Mice
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Active Induction of Autoimmune Ovarian Disease with a ZP3 Peptide

  Materials
  • Incomplete Freund's adjuvant (IFA; Sigma)
  • Mycobacterium tuberculosis H37RA (Difco)
  • Murine ZP3 peptides, synthesized and analyzed according to established methods (unit 9.7) stored as lyophilized powder in a dessicator or as 1 mM aliquots dissolved in H 2O at −70°C
  • Female B6AF1 mice, >6 weeks of age
  • Tribromoethanol (unit 1.4)
  • 50‐ml screw‐cap polypropylene tubes
  • 18‐G and 25‐G hypodermic needles
  • 3‐ml plastic or glass syringes with the Luer‐Lok tips (Becton Dickinson)
  • Female luer union (Rainin)
  • Additional reagents and equipment for anesthesia (unit 1.4) and injection (unit 1.6) of mice and immunohistochemistry (unit 21.4)

Support Protocol 1: Evaluation of Autoimmune Ovarian Disease

  Materials
  • Bouin's fixative (unit 15.16)
  • 70% ethanol
  • Hematoxylin and eosin stains (Fisher)
  • Surgical instruments
  • Instruments for embedding and sectioning (also see unit 21.4) including:
  •  Tissue processor (e.g., Tissue‐Tek)
  •  Embedding station (e.g., Tissue‐Tek)
  •  Microtome (e.g., Olympus)
  •  Microscopic slides (e.g., Fisher)
  •  Coverslips (Columbia Diagnostics)
  • Additional reagents and equipment for oophorectomy of mice ( protocol 2), and paraffin embedding and sectioning of tissue (unit 21.4)

Support Protocol 2: Adult Oophorectomy in Mice

  Materials
  • Adult female B6AF1 mice
  • Tribromoethanol (unit 1.4)
  • 70% ethanol
  • Sterile surgical instruments:
  •  Scissors, sharp blunt points (Miltex 5‐272)
  •  Scissors, sharp points (Miltex 5‐270)
  •  Toothed forceps (Miltex 6‐114)
  •  Pointed toothless forceps (Miltex 6‐118)
  •  Mosquito forceps, straight standard pattern (Miltex 7‐2)
  •  Small needle holder (Miltex 8‐40)
  • Sterile suture materials:
  •  4–0 silk (precut; without needle; Ethicon)
  •  4–0 silk (with reverse cutting needle; Ethicon)
  •  6–0 silk (with cutting needle; Ethicon)
  • HistoPrep biopsy bags (Fisher)
  • Additional reagents and equipment for anesthesia of rodents (unit 1.4)

Support Protocol 3: Neonatal Ovariectomy in Mice

  Materials
  • Neonatal or young female B6AF1 mice (24 hr to 7 days of age)
  • Gauze (4 × 4–in.)
  • 100 × 15–mm polystyrene petri dishes with lids (Fisher)
  • Dissecting microscope (Olympus)
  • Fiber optic cool light source (Dolan Jenner)
  • Sterile surgical instruments:
  •  4‐in. micro‐iris scissors, straight, sharp‐pointed (Miltex)
  •  Tweezers, length 110 mm, tip 0.13 × 0.08–mm (Roboz, pattern no. 4, RS‐4972)
  •  Tweezers, length 110 mm, tip 0.10 × 0.06–mm (Roboz, pattern no. 5, RS‐4976)
  •  8–0 or 9–0 black monofilament nylon sutures with 140‐ or 100‐µm needle,   respectively, on 135° curve taper (Sharpoint)

Support Protocol 4: Implantation of Ovarian Grafts in Mice

  Materials
  • Normal or treated 6‐ to 8‐week B6AF1 females as ovary donors and recipients
  • Serum‐free DMEM medium (Bio‐Whittaker)
  • 70% ethanol in a spray bottle
  • Tribromoethanol anesthesia (unit 1.4)
  • Sterile surgical instruments:
  •  4 3/4‐in. sharp/blunt point plastic surgery scissors (Miltex 5‐272)
  •  4 3/4‐in. delicate, serrated Adson dressing forceps (Miltex 6‐118, 2 pairs)
  •  4 3/4‐in. sharp/sharp point plastic surgery scissors (Miltex 5‐270)
  •  4‐in micro‐iris scissors, straight, sharp‐pointed (Miltex 18‐1618)
  •  Tweezers, length 110 mm, tip 0.13 × 0.08–mm (Roboz, pattern no. 4, RS‐4972)
  •  Hirschman jeweler‐type forceps, length 110 mm (Karl Storz, E1947 5H)
  •  43/4‐in. Hudson (Ewald) tissue forceps with 1 × 2 teeth (Milltex 6‐114)
  • Sterile Kimwipes
  • 4‐0 and 6‐0 silk sutures, sterile (Sharpoint)
  • Additional reagents and equipment for anesthesia of mice (unit 1.4)
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Figures

Videos

Literature Cited

Literature Cited
   Dean, J. 1992. Biology of mammalian fertilization: Role of the zona pellucida. J. Clin. Invest. 89:1055‐1059.
   Garza, K. and Tung, K.S.K. 1995. Frequency of T cell peptide crossreaction determined by experimental autoimmune disease and autoantibody induction. J. Immunol. 155:5444‐5448.
   Garza, K., Griggs, N.D., and Tung, K.S.K. 1997. Neonatal injection of an ovarian peptide induces autoimmune ovarian disease in female mice: Requirement of endogenous neonatal ovaries. Immunity 6:89‐96.
   Lou, Y.H., Ang, J., Thai, H., McElveen, F., and Tung, K.S.K. 1995a. A ZP3 peptide vaccine induces antibody and reversible infertility without ovarian pathology. J. Immunol. 155:2715‐2720.
   Lou, Y.H., McKelveen, F., Adams, S., and Tung, K.S.K. 1995b. Altered target organ: A mechanism of postrecovery resistance to murine autoimmune oophoritis. J. Immunol. 155:3667‐3673.
   Lou, Y.H., McElveen, F., Garza, K., and Tung, K.S.K. 1996. Rapid induction of autoantibodies by endogenous ovarian antigens and activated T cells: Implication in autoimmunity pathogenesis and B cell tolerance. J. Immunol. 156:3535‐3540.
   Rankin, T., Familiar, M., Lee, E., Ginsberg, A., Dwyer, N., Blanchette‐Mackie, J., Drago, J., Westhal, H., and Dean, J. 1996. Mice homozygous for an insertional mutation in the ZP3 gene lack a zona pellucida and are infertile. Development 122:2903‐2910.
   Rhim, S.H., Millar, S.E., Robey, F., Luo, A.M., Lou, Y.H., Yule, T., Allen, P., Dean, J., and Tung, S.K. 1992. Autoimmune disease of the ovary induced by a ZP3 peptide from the mouse zona pellucida. J. Clin. Invest. 89:28‐35.
   Tung, K.S.K., Lou, Y.H., Garza, K.M., and Teuscher, C. 1997. Autoimmune ovarian disease: Mechanism of disease induction and prevention. Curr. Opin. Immunol. 9:839‐845.
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