Technical Tips Online, Vol. 6, 2001

A faster procedure for preparingamniotic cells for sexing embryosRafael Jimenez, Francisco J. Barrionuevo and Miguel BurgosDepartamento de Genetica, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain

Keywords: Microscopy, Tissue culture

Most procedures in mammalian developmental biology

involve the use of embryos from mouse or another

species. In many cases, the sex of an embryo must be de-

termined rapidly before carrying out further procedures.

For instance, when several embryos of the same sex have

to be pooled for RNA purification in gene-expression

studies, or for tissue cultures. The time lag before the

embryo tissues are used in any of these techniques is crit-

ical for accurate experimental results. The PCR-based

procedures used to detect Y-linked DNA sequences or

chromosome preparations are time consuming and thus

clearly unsuitable for this purpose.

▼Palmer and Burgoyne (Ref. 1) developed a relatively fastmethod to prepare amniotic cells, enabling the sex of mouseembryos to be determined according to the presence or ab-sence of a sex-chromatin body. Although the entire pro-tocol requires ∼15–20 min, this technique has been usedfrequently (Ref. 2–4) because it was the only one availablefor the rapid sexing of embryos (Ref. 5). However, we havenow developed an alternative method to prepare amnioticcells that is based on the Meredith’s technique for preparingmeiotic chromosomes from mammalian testes (Ref. 6). Be-cause our protocol does not need to wait for centrifugationor air drying, sexing time is reduced to ∼5 min.

1. ProtocolRemove the extraembryonic membranes as a whole and

draw off the amniotic liquid by brief contact with apiece a filter paper. Place the membranes into a smallPetri dish containing 3:1 methanol:glacial acetic acidfixative. During fixation, the amnion, easily recognized

Corresponding author: rjimenez@ugr.es

by its veil-like appearance, is separated from the othermembranes. Fixation is complete in 1 min.

After fixation, lift a small piece of the amnion with finestraight forceps. Remove excess fixative by touchingseveral times with a clean, dry glass slide and transferthe amnion to the bottom of a 1.5 ml eppendorf tubecontaining a single drop (20–40 µl, depending on thesample size) of 60% glacial acetic acid. In this solution,cells will disaggregate in about 1 min. Repeated pipet-ting with a micropipette might reduce the time takenin this step.

Lift the cell suspension into a micropipette tip and placeit onto a clean glass slide that has been prewarmed to60◦C on a flat thermostatic plate. Immediately, drawthe cell suspension up from the plate without remov-ing the micropipette tip from the slide. The suspensionshould be placed successively onto different regionsof the slide and quickly removed until the suspensionevaporates or the slide is covered with small droplets.Amniotic cells will appear in concentric rings at theperiphery of the areas where the drop was placed. Anyresidual liquid will evaporate in few seconds at 60◦C.Alternatively, the slide can be immediately rinsed indistilled water and quickly dried by shaking or blow-ing.

Stain the cells by placing a drop of 1% aqueous toluidineblue onto the slide and covering with a coverslip. Theamniotic nuclei can be immediately examined undera light microscope to look for a sex-chromatin body,which takes less than 1–2 min.

The total time needed to sex an embryo is thus reduced toless than 5 min. Despite its simplicity, this method provideshigh-quality amniotic preparations. Most female cells havea dense chromatin body at the periphery of the nucleus(Fig. 1a), which is clearly distinguishable from the paler,centrally located, nucleoli. Most male cells lack any stainedspot other than the nucleoli (Fig. 1b).

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Technical Tips Online, Vol. 6, 2001 Technical Tips

Fig. 1. Amniotic cells from embryos of the mole species Talpa occidentalis prepared as described in the text. (a) Female cells showing positive staining forsex chromatin (arrows). (b) Male cells showing no sex chromatin body.

The total time spent sexing the embryo can be shorterthan the time required to dissect certain embryonic organsor tissues. While one worker is performing the dissection,someone else can make the preparations of sex chromatin,and the sex of the embryo can be identified before the dis-section ends without lengthening the time of the overallprocedure.

This peer-reviewed article can be cited as: Jimenez, R.(2000) A faster procedure for preparing amniotic cells forsexing embryos. Technical Tips Online T02062.

AcknowledgementsThis work was supported by the Spanish D.G.E.S. (projectPB96-1420) and by the Junta de Andalucıa (groupCVI109).

References1 Palmer, S.J. and Burgoyne, P.S. (1991) Development 113, 709–714.2 Capel, B. et al. (1996) Establishment and characterization of condition-

ally inmortalized cells from the mouse urogenital ridge J. Cell Sci. 109,899–909.

3 Capel, B. et al. (1999) Migration of mesonephric cells into the mammaliangonad depends on Sry Mech. Dev. 84, 127–131.

4 Merchant-Larios, H. and Moreno-Mendoza, N. (1998) Mesonephric stro-mal cells differentiate into Leydig cells in the mouse fetal testis Exp. CellRes. 244, 230–238.

5 Hogan, B. et al. (1994) Manipulating the Mouse Embryo, Cold SpringHarbor Laboratory Press 384.

6 Meredith, R. (1969) A simple method for preparing meiotic chromosomesfrom mammalian testis Chromosoma 26, 254–258.

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