838 



SPERM, OVA, AND PREGNANCY 



tions. Abstrictions of the polar bodies were 

 completed 45 minutes later. 



The interesting observations of Austin 

 (1951c) on the sequence of events during 

 formation of the second polar body in the 

 living rat ova deserve special mention. In 

 the unfertilized egg the chromosomes are 

 arranged on the metaphase plate with the 

 spindle lying paratangentially to the sur- 

 face, usually in close association with the 

 abstricted first polar body. Within a few 

 minutes after the sperm head has penetrated 

 the vitellus, and before it shows any de- 

 tectable change, the chromosomes on the 

 second maturation spindle pass to anaphase. 

 The telophase stage is reached about 75 min- 

 utes after the initial penetration by the 

 sperm. Then, there is a 20-minute period dur- 

 ing which no further change is noted. Subse- 

 quently, the spindle slowly moves away from 

 the surface and begins to rotate in such a 

 way that its final position is at right angles 

 to its original location. Rotation is com- 

 pleted in about 50 minutes. The spindle then 

 elongates and becomes narrower, the process 

 terminating in abstriction of a clear vesicle 

 containing the clumped chromosomes. Since 

 it was necessary to flatten the egg consider- 

 ably in order to be able to observe the spin- 

 dle under the phase microscope, complete 

 abstriction of the polar body did not occur. 



Similar observations on the formation of 

 second polar bodies in rat ova were re]iorted 

 by Odor and Blandau (1951). Approxi- 

 mately 2000 eggs were removed at varying 

 intervals after ovulation and sperm penetra- 

 tion. The eggs were examined either in the 

 fresh condition or after histologic prepara- 

 tion. In the majority of ova, the second polar 

 body had been abstricted completely by the 

 end of the 4th hour after semination. 



H. PRONUCLEI FORMATIOX, SYNGAMY, AND FIRST 

 SEGMENTATION DIVISION 



As mentioned earlier, the general concept 

 of the mechanism of fertilization in mam- 

 mals has been based almost entirely on the 

 examination of fixed and stained material. 

 Even so, it is remarkable that a story of con- 

 tinuing development should have evolved by 

 the piecing together of evidence from killed 

 eggs, the age of which could not be deter- 

 mined within narrow limits. The more recent 



advances involving an evaluation of the 

 temporal relationship between ovulation 

 and the various phenomena of fertilization 

 may be said to be due largely to the applica- 

 tion of phase contrast microscopy to the 

 studies of living rat ova (Austin and Smiles, 

 1948; Odor and Blandau, 1951; Austin, 

 1951a, b, 1952a; Blandau and Odor, 1952; 

 Austin and Braden, 1954a, b). 



Employing this method, Austin and Smiles 

 observed fertilized eggs that were obtained 

 by inducing ovulation in immature rats by 

 means of gonadotrophins and subsequently 

 allowing the females to mate. The recovered 

 zygotes were kept at body temperature and 

 development was followed continuously with 

 the phase microscope. The details of the fer- 

 tilization process described by Odor and 

 Blandau were the result of examining sev- 

 eral thousand living and fixed fertilized eggs 

 recovered from sexually mature females at 

 specific time intervals after ovulation and 

 fertilization. 



In the rat the complete process of fertiliza- 

 tion, from the penetration of the ooplasm by 

 sperm until the first segmentation division, 

 requires approximately 24 hours. In general, 

 the first 8 hours after sperm penetration is 

 the period of the formation of the second 

 polar body and the initial development of 

 the male and female pronuclei (Fig. 14.12). 



Changes in the morphology of the living 

 sperm head can be noted as early as 10 min- 

 utes after penetration of the ooplasm and 

 involve a loss of sharpness of outline and 

 contrast, first in the posterior and caudal re- 

 gions of the head. The decrease in contrast 

 continues until finally the whole nuclear part 

 is almost invisible in the living specimen, 

 even under the phase-contrast objectives 

 (Fig. 14.12, Jf). Concomitantly the head in- 

 creases greatly in size and fluidity. During 

 the initial period of swelling of the nuclear 

 portion, the bifid perforatorium becomes de- 

 tached (Fig. 14.12, 3). Approximately 2 

 hours after the sperm has entered, the j^ri- 

 mary nucleoli make their appearance within 

 the enlarged sperm nucleus. Time-lapse cine- 

 microphotography has shown that the nu- 

 cleoli enlarge by the fusion of minute nucleo- 

 lar aggregations. The larger nucleoli then 

 fuse one with another until only a single 

 large nucleolus is present (Fig. 14.13, 1 and 



