Neonatal Mouse Ovary Isolation: A Surgical Procedure to Harvest Pair of Ovaries from Neonatal Mouse Model

Published: April 30, 2023

Abstract

Source: et al. Morgan, S. Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles. J. Vis. Exp. (2015).

In this video, we describe a surgical procedure to isolate ovaries from a neonatal mouse.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 

1. Neonatal Ovary Dissection and Culture

  1. Place 1 mL of dissection medium (dissolve 3 mg/mL BSA in Leibowitz L15 medium and filter sterilize through a 0.2 µm pore, 25 mm diameter filter) into each sterile glass embryo dish.
  2. Cull neonatal mouse pups aged between postnatal day 0 and 5, culling by decapitation.
  3. Grasp the skin covering the abdominal wall using a pair of fine dissection forceps and make a large incision in the skin and body wall. Pull open the incision so the entire abdomen is exposed. The bladder is usually engorged at this stage and can be punctured to make dissection easier.
  4. Move the guts out of the way using watchmaker forceps. Follow the uterine horns from the bladder up to the kidney on each side. The ovary is located just below the kidney at the top of the uterus and will appear as a cloud-like structure under a dissecting microscope.
  5. Grasp the ovary gently with watchmaker forceps and use scissors to sever its attachment to the uterus. Transfer the pair of ovaries into embryo dishes containing pre-warmed dissection medium.
  6. Carry out fine dissection of ovaries under a dissection microscope on a heated stage (37 °C). Use insulin needles to trim away the bursal sac and any excess material including the fallopian tube, until only the ovary remains.
  7. Transfer each ovary into the well of a culture plate using a finely drawn curved glass pipette.

Disclosures

The authors have nothing to disclose.

Materials

Leibowitz L15 Invitrogen 11415049
αMEM Invitrogen 22571020 Supplied as sodium bicarbonate buffered (2,200 mg/L)
Bovine serum albumin Sigma Aldrich A9418 For dissection medium
Bovine serum albumin Sigma Aldrich A3311 For culture medium
Bovine serum albumin Sigma Aldrich A2153 For coating glass pipettes
FSH Merck Serono Gonal F Batch testing is often necessary. Make up stock solution of 1IU/10µL in αMEM and store at -20 °C
Syringe filters (25 mm, 0.2 µm)  Greiner  16532K  Cellulose acetate filter for filtering larger (>5 mL) volumes of medium.
Sterile tubes Greiner 187261
24-well plate Greiner 662160
Insulin needles BD medical supplies 037-7606 0.33 mm (29 G) + 1 mL
Glass embryo dishes VWR 720-0579 Sterilize, then warm before use
Glass pipettes Fisher Scientific 10209381 BSA coated before use
Acupuncture needles Acumedic LTD 30 mm x 0.25 Type C
Formalin solution, neutral buffered, 10% Sigma Aldrich HT5014-120mL
Whatman nucleopore membranes Camlab WN/110414 Use shiny surface up, polycarbonate, 13 mm diameter, 8.0 µm pore size

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Cite This Article
Neonatal Mouse Ovary Isolation: A Surgical Procedure to Harvest Pair of Ovaries from Neonatal Mouse Model. J. Vis. Exp. (Pending Publication), e20756, doi: (2023).

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