450 MOUSE EMBRYO 



check may be made (until one has had experience in identifying the plug) by 

 means of a vaginal smear. This is done by inserting a few drops of physio- 

 logical saline into the vagina with tapered pipette tip, sucking it out and ex- 

 amining it for spermatozoa. The females with plugs (or sperm) should be 

 segregated and labelled for time of mating. 

 4. To calculate the time of conception: Since this can occur at any time that 

 the sexes are together, but under the above conditions is limited to from 

 5 P. M. until 9 A. M. , and is most likely to occur around midnight, the range 

 of error is, at most, a matter of a few hours. Thus, with some variability 

 most evident only during the earliest development, females with vaginal 

 plugs at 9 A. M. may be labelled as 0. 5 day pregnant. The mating period 

 for any female is short, and occurs every 5 days, so that one could hardly 

 expect more than 10% successful copulations under the above conditions. 

 Thus, to obtain 10 simultaneous matings one would have to expose over 100 

 females simultaneously to an adequate number of males. On five successive 

 night matings with the same males and females, one often finds certain 

 nights most productive. 



At stated times after conception, embryonic material may be observed in the following 

 manner: Kill the female by cervical dislocation. This is done by grasping the neck just 

 behind the head with thumb and forefinger of one hand, and quickly pulling (jerk) on the 

 tail with the other hand. This is the most quick and considerate method of killing a mouse. 

 The bicornuate uterus and attached ovaries may be quickly dissected out, with minimum 

 handling, and placed upon a piece of white filing card to which it will stick. Immerse in 

 Bouin's (or other fixative) after labelling the card in pencil as to exactly what stage of 

 development (and experimental conditon) is involved. After several hours gently remove 

 the fixed uteri from the card, but leave in Bouin's for 24-48 hours. Later stages of de- 

 velopment will require longer fixation. Embryos older than 9. 5 days should be exposed 

 by opening the uterus and piercing the membranes to allow penetration of the fixative. 

 (See Rugh' s revised edition 1961: "Laboratory Manual of Vertebrate Embryology" for the 

 normal embryonic stages of the mouse. See also Austin & Smiles 1948: Jour. Roy. Micr. 

 Soc. 68:13). It is also possible to flush eggs and/or early embryos from the oviducts or 

 uteri, prior to implantation at 4. 6 days, and observe them in physiological saline in toto. 



GENERAL OUTLINE OF DEVELOPMENTAL STAGES OF THE MOUSE:* 



1. Fertilization in upper oviduct, generally monospermic. 



2. First cleavage at 24 hours, in upper oviduct. 



3. Second cleavage at 36 hours, in upper oviduct. Holoblastic. 



4. Sixteen cell morula at 2. 5 days, free in uterus. 



5. Blastula stage (32+ cells) at 3. 5 days, free in uterus. 



6. Implantation stage at 4. 5 days, spaced in uteri. 



7. Germ cell layers differentiated at 4. 5 to 5. days. 



8. Egg cylinder stage at 6 to 6. 5 days. 



9. Embryonic and extra-embryonic regions distinguished at 7. to 8. days. Head 

 process, primitive streak, mesodermal cavities, active neurogenesis, extra- 

 embryonic membranes including amnion which extends over the entire dorsal 

 surface, and the first definitive somites appear. Embryo early "C" shape. 



10. Heart formed and circulation starts at 9. days. Tissue differentiation active, 

 as in all mammalian embryos. 



Artificial insemination as well as the transplantation of fertile and non-fertile ova is 

 possible in the mouse (see McLaren & Biggers '58) for specific research purposes. 



* Rot data are so close to the mouse that substitution is possible, except that more space, food and water per animal are required. 



