MAMMALIAN OVARY 



473 



sensitive than tliose in which the estrogen is 

 administered subcutaneously. When the 

 mean number of arrested mitoses was used 

 as tlie measure of estrogenic activity, the 

 sensitivity was increased another ten times 

 ( Martin and Claringbold, 1958). 



Immature rats or mice can also be used 

 foi' the assay of estrogens. The establish- 

 ment of vaginal patency and mucified or 

 cornificd vaginal smear denote estrogen ef- 

 fects. Vaginal patency per se, however, is 

 not specific for estrogen because androgen 

 will also induce precocious vaginal patency 

 (Rubinstein, Abarbanel and Nader, 1938; 

 Marx and Bradbury, 1940). Injection of es- 

 trogen into immature animals causes a rapid 

 increase in the weight of the uterus, due to 

 water imbibition. Astwood (1938) found 

 that the immature rat uterus increases in 

 weight as early as 6 hours after an injection 

 of estrogen; however, the optimal response 

 was at 30 hours. Just above threshold levels 

 graded doses produce graded increases in 

 uterine weight. This makes it possible to 

 plot a dose-response curve so that closer 

 approximations of potency can be achieved. 

 Some authors have used ovariectoraized ani- 

 mals for the uterine response method. This 

 requires a prior operation and subsequent 

 adhesions may make it difficult to strip out 

 the uterus cleanly at the end of the test. 



Whatever the test, each estrogen deriva- 

 tive or synthetic estrogen has an optimal 

 assay interval for maximal effect depending 

 in part on its solubility, rate of absorption, 

 and utilization (Hisaw, 1959). For this rea- 

 son a standard assay may not be an accurate 

 indicator of the estrogenic potency of sev- 

 eral comijounds. This factor plus some 

 competitive antagonism make this method 

 impractical for the assay of mixtures of es- 

 trogens (Merrill, 1958). Whatever their 

 faults, the bioassay methods in general are 

 very sensitive and will detect estrogens in 

 0.1 to 1.0 ixg. quantities. 



The chemical assay methods for estrogen 

 are rather involved and have usually been 

 relial)le only in milligram quantities. Fluo- 

 rometric methods were tried and generally 

 discarded because frequently small amounts 

 of contaminants were strongly fluorescent; 

 consequently fluorescence was being ob- 

 tained in the absence of biologic activity 



(Bitman, Wrenn and Sykes, 1958) . Recently 

 paper chromatographic methods have per- 

 mitted sufficient purification and isolation 

 to make identification and quantification of 

 estrogens in microgram quantities (Brown, 

 1955; Smith, 1960; Svendsen, 1960). 



The most common bioassay methods for 

 progesterone are still the Corner-Allen and 

 the Clauberg tests which utilize the rabbit. 

 The animal is primed with estrogen and 

 then given progesterone after an appropriate 

 interval. A portion of the uterus is removed 

 and examined histologically for the degree 

 of glandular develojiment in the endome- 

 trium. The test is relatively insensitive since 

 it requires about 1 mg. progesterone per rab- 

 bit. McGinty, Anderson and McCullough 

 (1939) increased the sensitivity of the test 

 to 0.5 to 5.0 /xg. by injecting the progesterone 

 into the lumen of an isolated segment of the 

 rabbit uterus. The histologic response of the 

 endometrium in this isolated segment was 

 then judged. 



Hooker and Forbes (1947) adapted the 

 McGinty intra-uterine technique to the 

 uterus of the ovariectomized mouse. The 

 end result is judged histologically by the 

 characteristics of the endometrial stromal 

 nuclei of the isolated uterine segment. The 

 sensitivity is of the order of 0.3 fxg. per ml. 

 and the method has been used widely. This 

 advantage of the Hooker-Forbes technique 

 is that it is sensitive enough to detect ges- 

 tagens in l)lood plasma and liquor folliculi. 

 Disadvantages are that the test is not spe- 

 cific for progesterone, and that certain ges- 

 tagens such as 17-a-hydroxyprogesterone 

 which is devoid of progestational activity 

 in some species (rabbit, guinea pig, man) 

 are very active in the mouse test (Zarrow, 

 Neher, Lazo-Wasem and Salhanick, 1957; 

 Short, 1960). 



There are spectrophotometric techniques 

 for jn'ogesterone assay (Reynolds and Gins- 

 burg, 1942; Zander and Simmer, 1954; 

 Short, 1958; Sommerville and Deshpande, 

 1958 ) . These methods have the advantage of 

 instrumental precision, but require rather 

 tedious initial chemical purification. How- 

 ever, they have proved of especial value 

 in comparative studies of the blood levels 

 of progesterone in sheep with active and 

 inactive ovaries, in studies of the progester- 



