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

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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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