HYPOPHYSECTOMY AND EARLY AMPHIBIAN DEVELOPMENT 



PURPOSE : By means of surgical extirpation to determine the relation of the epithelial 

 hypophysis to early amphlhian development. 



MATERIAI^ : 



Biological : Early tail-bud stages of any amphiblEin (Anura stage #l8, Urodela stage 



#29). 



Technical : Standard equipment. 



METHOD : 



Precautions : 



a. Use only embryos that are not injured during removal from their Jelly capsules. 



b. Avoid bacterial contamination following the operation. If this becomes a factor, 

 operate in 0.1^ sodium sulfadiazine in Standard Solution (or Operating Medium for 

 Urodeles) . 



c. Endeavor to remove the hypophysis completely, but no other tissue. (The success 

 of this operation can be tested only by subsequent histological examination. ) 



Control : The control for this experiment consists of surgical cutting in the vicinity 

 of the hypophysis, but without removal of any cells. 



Procedure : 



a. Bemove all coverings, including the vitelline (fertilization) membrane, from a 

 group of embryos at the appropriate stage (see above). Place them in 2X Standard 

 Solution or in Spring Water (Anura) or in Urodele Operating Medium (Urodeles) 

 over a base of agar. The agar will prevent adhesion of the epidermis to the 

 glass bottom of the dish. 



b. Prepare an operating dish with base of soft paraffin or of Permoplast, and, with 

 a ball tip, mould a depression so that the embryo can be held securely with its 

 face looking upward toward the operator. 



c. Locate the hypophyseal groove (from stomodeum to hypophysis) and its dorsal hypo- 

 physeal pit. This is the region of ingrowing of the pigmented, ectodermal cells 

 which constitute the hypophysis. Use a double-edged lancet, or micro- (glass) 

 needles to remove the wedge of pigmented hypophysis. This anlage grows inward 

 between the roof of the pharynx and the floor of the brain ( Infundibulum) but 

 neither of these other tissue areas should be disturbed, if possible. With small 

 hair loop excavate all pigmented cells from the hypophyseal pit.* 



d. Leave the embryo in operating medium until the wound heala, about 50 minutes, 

 then return it to the normal culture medium (i.e., Standard for Anura and Growing 

 Medioun for Urodeles). Keep operated embryos separately in #2 Stenders, with agar 

 bases, preferably at temperatures slightly below that of the laboratory. 



OBSERVATIONS AND TABULATION OF DATA : 



a. Make sketches immediately after the operation and at 15 minute intervals during 

 the healing process of the hypophyseal area. Healing should be complete within 

 1 hour . 



b. At weekly intervals after the operation make drawings (or take photographs) of 

 the operated and control embryos, side-by-side, to show any 



1. Changes in pigmentation 



2. Differences in rate of development (i.e., size differences) 

 Eemember that these embryos must be fed after the stage #1+2. 



c. Embryos which show prono\inced effects of hypophysectoipy (silvery appearance), 

 stunting, etc.) should be sectioned (transversely and sagl tally) to determine the 

 extent or success of the hypophyseal extirpation. Control embryos of the same 

 age should be sectioned for direct comparison. Note also any variations in the 

 development of the thyroid glands. 



* 



See section on'^Thyroidectoiny and Early Development" for photogi-aph of sagittal section of 

 Anuran embryo showing hypophysis. 



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