20. ARTIFICIAL PARTHENOGENESIS* 



PURPOSE: To repeat the earlier experiments, using modern methods of inducing ovula- 

 tion and of equipment, in an attempt to initiate the development of the amphibian egg 

 by artificial means (i. e. , without benefit of spermatozoa). 



MATERIALS: 



Biological: Uterine eggs from an ovulating anuran: Rana or Bufo. Blood from a 

 second non-ovulating female anuran, same species. 



Technical : Slides, Petri dishes, finger bowls, #Z Stenders, moist chamber, sec- 

 tion lifter, sharp-pointed (3 to 9 (i) glass or platinum (20 to 30 p.) nee- 

 dles, and china marking pencil. 



METHOD: 



Precautions : 



1. All articles and female frogs must be kept sperm-sterile. The instruments 

 and glassware may be boiled for 5 minutes or immersed in 70% alcohol and 

 air dried. 



2. The female frog which produces the eggs for the experiment should be iso- 

 lated from all males for several days prior to the experiment and should be 

 washed off with tap water and dried before stripping. 



Controls : Two types of controls are necessary for this experiment. 



1. Some eggs are to remain untreated, but should be placed side-by-side with 

 the eggs experimentally treated. This provides identical environmental con- 

 ditions for the experimentals and the controls, with but a single variable. 



2. The eggs should be tested to determine whether they are in fertilizable con- 

 dition. Following the conclusion of the experiment, some of the uterine eggs 

 should be normally inseminated by frog spermatozoa, of the same species, 

 in another laboratory. There must be no possible contamination of the ex- 

 perimental eggs with spermatozoa. 



Procedure : 



1. Adjust a low-power microscope so that the heat-absorbed light will strike 

 the eggs from a 45° angle from above. A lantern slide cover glass might be 

 used to protect the microscope stage from water. 



2. Place 10 clean microscope slides, a slight distance apart, on clean paper 

 towelling. On the upper left hand corner of each mark "C" (for controls) and 

 below on the lower left hand corner of each slide mark "X" (for experimen- 

 tals), using a china marking pencil. Number the slides in sequence. 



3. Strip a single row of eggs from the uteri of an ovulating female, placing 

 them along the length of the slide opposite "C" and then another opposite "X". 

 Try to strip eggs in a single row so that they will not lie over each other, 

 and will adhere to the slide. Place all 10 slides of eggs in a moist chamber 

 where they may remain for an hour or more without deleterious effects. 



(The chamber should have stood for at least an hour so that the contained air 

 is completely saturated with water vapor. ) Such eggs will lose their CO2 

 and thereby facilitate their physiological maturation (Bataillon & Tchou-Su, 

 1930). 



4. Pith a non-ovulating female frog; lay it on some paper towelling; cut through 

 the leg muscles to prevent further reflex movements; open the abdomen and 



* This laboratory procedure has been organized with the very generous help of Dr. C. L. Parmenter. The results could also be 

 called "gynogenetic haploidy. " 



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