BIOLOGY OF EGGS AND IMPLANTATION 



859 



fill interplay between the embryos and cor- 

 nua as described for the rabbit. The blas- 

 tocysts of these rodents are definitely 

 polarized in relation to the uterine epithe- 

 lium at the time of attachment and in- 

 vasion. Although it is universally stated 

 that the blastocyst does not have the ca- 

 l^acity for independent movement in utero, 

 observations on the behavior of the guinea 

 pig blastocyst in tissue culture and the 

 cytologic descriptions of the attachment 

 cones in the monkey, ground squirrels, and 

 chipmunks suggest that the blastocyst plays 

 an active role in its positioning at the time 

 of attachment. This possibility would en- 

 courage one to examine more carefully 

 the living blastocysts of various animals 

 at the time of attachment. From some of 

 the earlier investigations, it would seem 

 that the expansion of the rabbit blastocyst 

 is dependent on physiologic factors external 

 to the egg itself (Pincus and Werthessen, 

 1938). Thus, blastocyst expansion is inter- 

 fered with if ovariectomy is performed or 

 estrogen is injected 3 to 5 days after mat- 

 ing. On the other hand, injections of pro- 

 gesterone can reverse the effect of estrogen 

 (Burdick and Pincus, 1935; Pincus, 1936; 

 Pincus and Kirsch, 1936). Allen and Corner 

 (1929) showed that if progesterone is in- 

 jected into rabbits ovariectomized shortly 

 after fertilization, the fertilized eggs will 

 implant normally. If fertilized rabbit ova 

 are grown in watch glass cultures, they will ■ 

 cleave normally, but they herniate and col- 

 lapse during the blastocyst stage (Lewis 

 and Gregory, 1929) . If crystalline progester- 

 one is added to these cultures, there is no 

 increase in the rate of cleavage nor is 

 herniation or collapse prevented (Pincus 

 and Werthessen, 1937). The same investi- 

 gators have shown that regular expansion 

 of the blastocyst is obtained if the morulae 

 or blastocysts are cultured in homologous 

 serum and the medium is continually circu- 

 lated. Recently, Bishop observed that ex- 

 pansion is suppressed if the oviducts of 

 rabbits are ligated soon after the blastocysts 

 have entered the uterus. The implication 

 is that some oviducal factor is necessary for 

 expansion. The problem is complicated by 

 the fact that the egg does not expand dur- 

 ing its 3 day sojourn in the oviduct. 



From the observations recorded above, 

 it seems that in order to stimulate normal 

 growth and expansion of the blastocyst, in 

 the leporid family of rodents at least, pro- 

 gesterone must act in some way on oviducal 

 and uterine metabolism since both parts 

 of the genital tract are probably involved. 



The specific physicochemical processes 

 in blastocyst expansion are not known. A 

 plausible explanation is that the expansion 

 may be due simply to the processes of 

 osmosis, the changes in size being related 

 to ionic variations of the fluid within the 

 blastocyst cavity and the surrounding en- 

 vironment. It is more likely, however, that 

 comjMex processes of active transport are 

 involved, and, if these are to be elucidated, 

 help from the biochemist and physical 

 chemist is essential. 



One of the difficulties confronting in- 

 vestigators so trained is the small amount 

 of material obtainable for study by the 

 conventional chemical methods. This is par- 

 ticularly true in such laboratory animals as 

 the mouse, rat, hamster, guinea pig, and 

 monkey, in which the blastocyst undergoes 

 very little expansion before implantation 

 and in which uterine secretions are present 

 in very minute amounts. Nevertheless the 

 recent approaches to the study of embryo 

 attachment and implantation in the rabbit, 

 particularly those by Boving (1954), Ben- 

 nett (1956) and Lutwak-Mann (1959), 

 offer a methodological approach that is 

 essential if the dynamic aspects of nidation 

 are to be understood. Lutwak-Mann espe- 

 cially and her co-workers have been the 

 most active in discerning the practical prob- 

 lems in the handling of early embryologic 

 material for biochemical study and in de- 

 vising sound methodologic approaches. 



In 1938 Pincus and Werthessen described 

 a crystalline deposit in the abembryonal 

 membranes of certain blastocysts of rabbits 

 removed on the 5th day after mating from 

 females which had been ovariectomized 18 

 to 20 hours after copulation. Boving (1954) 

 identified this crystalline material as cal- 

 cium carbonate and noted that there is 

 little or none present 3 to 4 days after 

 mating, but that the deposit increases to a 

 maximum at the 6th day post coitum. He 

 suggested that the osmotic effect of the 



