Inducing egg laying via hCG injection (Vize lab)

Protocol submitted by Peter Vize [1]

Both Xenopus laevis and tropicalis respond to stimulation with human chorionic gonadotropin (hCG) by laying eggs. This protocol focuses on laevis. A detailed protocol, including lots of images on animal restraint techniques, for tropicalis is available on the Harland laboratory web site [2]. Not many frogs respond to hCG in this manner and it is one of the big advantages of Xenopus. If you want an alternative species for comparative studies, Bombina orientalis also responds well to hCG.

hCG

hCG is purchased from Sigma, catalog number CG10, 10,000 Units per vial. The hormone is provided lyophilized and must be reconstituted before use. This is done by drawing 10 ml of sterile water into a syringe through a 23 to 26 gauge needle and gently pushing the tip of the needle through the rubber end cap. A second needle, not attached to a syringe, is also pushed through the end cap to act as a vent. The 10 ml of water is then gently pushed into the vial where it will rapidly dissolve the hormone in response to gentle shaking. Do not force the water in rapidly or agitate in such a way as to generate bubbles as this will denature the protein based hormone.

Remove the venting needle.

Once dissolved take your first sample by inverting the vial so the tip of the needle is immersed in the solution, pull back on the plunger until the desired volume has been drawn into the syringe.

Freeze the remaining hormone at minus 20 deg C. When you want to use more hormone thaw at room temp or in luke warm water, collect the required volume, and return the stock to minus 20 immediately.

hCG dosage

The amount of hCG to induce egg laying varies with season, frogs, frog size, age/activity of the hormone, time in which you want a response, and room temperature.

To keep things simple we keep all variables except one constant. Injected females are full sized, so approximately 12 cm in length. We inject at the same time, 6:00 p.m. (more or less) and always keep the frogs at 18 deg C in the dark until morning. Our only variable is hormone dosage, and this changes a little with season, frog size etc.

A recommended starting dose is 500 units per animal, so 500 ul volume of the 1,000 units/ml stock. If frogs do not lay at all, or lay too late the following day, select a new frog and try 600 units. We never need to use more than 700 units.

Some people like to pre-prime frogs, by giving them a low dose, often 50 to 100 units, then resting them for a week before priming. In you pre-prime you can use less priming hormone, or the same amount (500U) and get egg-laying in a much shorter time.

We typically inject three frogs, and on average two will respond in a usable time window.

Animal Restraint and Injection

There is a great series of photos and a detailed description of different techniques on the Harland laboratory web site[3]. We use the 'iron claw' technique, to use their nomenclature. The frog is held with its head facing towards your wrist and back legs poking forward between your fingers. Hold on firmly as the frog will be slippery, plus a firm grip helps to calm them. Slip the hypodermic syringe between the skin and the body muscle, inserting the tip just posterior to the lateral line and pushing it dorsally towards the head. Insert the needle about 1 cm forward from the puncture site in between the skin and the muscle. As you gently expel the hormone solution you will see the skin rise a little and a tiny amount may leak out of the needle entry site. There should be little or no blood at the puncture site. Once injected, pull the hypodermic out in the opposite direction to that used for insertion. Once again you should not enlarge the injection hole or cause any damage or bleeding when doing this. Return the frog to its bucket but keep a firm grip until the frog is actually in the water- if you soften your grip it will sense this and take off!

Recovery after egg laying

We let frogs rest after egg laying at 18 deg C overnight. Any residual egg laying will occur in this window and save fouling your frog tanks with unwanted eggs. After the 24 hour rest frogs are returned to the main holding system. They are rested for a minimum of three months before being primed for laying again.

If frogs are rested too long they may not lay well the next time they are primed. If this occurs simple note which frogs displayed this response, rest them for three months, and prime again- they should be good to go.