184 



HYPOPHYSIS AND GONADOTROPHIC HORMONES 



respectively, raises the question as to 

 whether or not the hormones themselves are 

 responsible for the staining reactions. The 

 parallelism between depth of staining and 

 hormone content does not contribute to the 

 solution of this problem, because this par- 

 allelism is a consecjuence of the fact that 

 the hormone is stored in the specific gran- 

 ules and the number of granules present de- 

 termines the depth of staining. This cjues- 

 tion, therefore, resolves itself into two parts. 

 First, are the hormones glycoproteins, and 

 second, are the hormones major constituents 

 of the granule contents? 



The reported sugar contents of the prepa- 

 rations of thyrotrophin, FSH, and LH pro- 

 duced by the fractionation of extracts from 

 the anterior lobes of domestic animals do 

 not establish the glycoprotein nature of 

 the hormones, because it has never been 

 shown that similar glycoprotein-containing 

 fractions do not result from the fractiona- 

 tion of extracts of tissue which are free from 

 these hormones. Taking advantage of the 

 ease with which rat hypophyses can be ob- 

 tained free from these particular hormones 

 by pretreatment of the animal with thy- 

 roxine and estrogen, I have compared the 

 extracts from hormone-free anterior lobes 

 with those from normal anterior lobes. Simi- 

 lar amounts of protein with similar sugar 

 contents were extracted from both types of 

 gland. Fractionations by salt or alcohol 

 which resulted in potent preparations of 

 thyrotrophin, FSH, and LH, when applied 

 to the extracts of the normal gland, gave a 

 similar partition of the glycoproteins ex- 

 tracted from the hormone-free gland. It is 

 clear that the preparations of the reputedly 

 glycoprotein hormones obtained by frac- 

 tionation of glandular extracts with varying 

 concentrations of salts or alcohol are com- 

 posed mainly of inert materials resulting 

 from the fractionation of a mixture of 

 plasma proteins and soluble cytoj^lasmic 

 constituents. 



In extracts from castrate rat anterior 

 lobes in which the gonadotrophins are pres- 

 ent at many times the normal level, the 

 FSH fraction contains a small but definitely 

 increased amount of PAS reacting material 

 compared with the corresponding fraction 

 from an equal weight of hormone-free an- 

 terior lobe. There is. therefore, in the l)as()- 



phil granules a glycoprotein which accom- 

 panies the FSH during fractionation of the 

 extract, and this glycoprotein is present in 

 amounts which would contribute to the 

 staining reactions of the granules. The po- 

 tency of this material is close to that of the 

 highly purified FSH preparations reported 

 by Steelman, Kelly, Segaloff and Weber 

 (1956). 



Persons observing pars anterior cells un- 

 der the microscope often form impressions 

 which exaggerate the amount of specific 

 granulation present in the pars anterior. 

 They often also have exaggerated ideas 

 about the potency of the separated hor- 

 mones. Taking account of the small per- 

 centage of granulated thyrotrophs in the 

 rat pars anterior and the small fraction of 

 the cytoplasmic content of these cells which 

 is in the form of granules, there must be 

 only about 1 /xg. of specific thyrotroph 

 granulation per mg. dry weight of anterior 

 lobe. Yet the anterior lobe has a potency 

 of 0.2 I.U. thyrotrophin per mg. dry tissue, 

 equivalent to 10 /xg. of the potent prepara- 

 tion of thyrotrophin obtained by Condliffe 

 and Bates (reported by Sober and Peterson, 

 1958). 



It seems reasonable to assume that baso- 

 phil granules, like other specific secretory 

 granules, contain a mixture of substances, 

 and it appears likely that, at least in the 

 rat pars anterior, the glycoprotein hormones 

 form a considerable proportion of the gran- 

 ule content and contribute significantly to 

 its staining reactions. 



E. DIFFERENTIAL STAINING OF BASOPHIL CELLS 

 BY RESORCIN-FUCHSIN, KRESOFUCHSIN, AL- 

 DEHYDE-FUCHSIN, AND ALCIAN BLUE: ^- 

 CELLS AND 8-CELLS 



The three basoi)hil cell types in the pars 

 anterior of some niainmals can be distin- 

 guished from each otlici' by diagnostic fea- 

 tures which are independent of any specific 

 diffei-ential staining of the granules, but 

 e\-en in these species a differential staining 

 of the sjiecific granulation is an important 

 aid in the study of specific types. In 

 some species the granules of different cell 

 types react differently to acid dyes and ap- 

 pear in different colors after trichrome stain- 

 ing methods. The recognition of the pres- 

 ence of a multiplicity of basophil types was 



