female vertebrates arc related to maturity and can 

 be studied by biofhemical and serological teclini(iues. 

 Serum vitellin has been associated with maturation 

 in hens (Roepke and Hughes, 1935) and related 

 serologically to egp; vitellin in fowl (Roepke and 

 Bushncll, 1936). Analogous conditions have been 

 described in other oviparous vertebrate classes in- 

 cluding teleosts. Uhlenluith and Kodama (1914), 

 as quoted by Sasaki (1932), used antisora prepared 

 from carp ovaries to distinguish serum of mature 

 female carp from serum of immature female and 

 male carp. Kidgway associated a serologically 

 detected serum factor of the sockeye salmon (also 

 found at high concentrations in the egg) with ma- 

 turity in females (Ridgway, Klontz, and Matsumoto, 

 1902); subsequently he has found the antiserum to 

 cross-react with mature females in all salmonid 

 species tested (Ridgway, unpublished data). Con- 

 currently, Vanstone and Ho (19G1), in studies of 

 electrophoretic i)atteins of coho salmon sera at vari- 

 ous stages of development, observed a component 

 that was characteristic of maturing females. Fine 

 and i:)rilhon (1903) identified a similar protein in 

 Salmo salar by immunodiffusion. Existing evidence 

 indicates that serum vitellin may be accounted for 

 by the following secpience of events in oviparous 

 vertebrates: under the control of the pituitary, 

 estrogen produced in the ovary stimulates produc- 

 tion by the liver of proteins that are passed through 

 the blood to the ovary and there utilized in yolk 

 formation. 



The present study attempts to unite the biological 

 and serological approaches in investigations of the 

 maturity of two pleuronected species. We origi- 

 nally intended to study only Pacific halibut. When 

 we found that collecting an adequate halibut sample 

 was impractical, we included English sole {Parophri/s 

 rrlvlus), a more available species. The study is 

 based on a serum-vitellin component, called the HM 

 factor, which occiu-s in mature female flounders. 

 The objective is to demonstrate that serological 

 methods may be advantageously applied in matur- 

 ity studies of these species by showing the relation 

 of the factor to various biological features. 



METHODS AND MATERIALS 



The serological mctiiods, preparation of the anti- 

 genic substance used in the methods, and the collec- 

 tion of samples for analysis as well as for deter- 

 mination of age of fish from which samples were 

 taken are described. 



SEROLOGICAL METHODS 



The procedure for the detection of the TIM factor 

 was a microslide adaptation of the Ouchterlony 

 method of double-diffusion prccijjitin analysis as 

 described by Ridgway et al. (1902). The diffusion 

 method provides a means of identifying antigenic 

 components of a solution through diffusion of the 

 solution and an antiserum towards one another in a 

 .semisolid medium,'' If the antiserum contains anti- 

 bodies specific for components of the solution, a 

 precipitate line is formed in the zone where given 

 antigen molecules meet specific antibodies in optimal 

 proportions. If two antigen solutions that are 

 placed adjacently diffuse towards a single antiserum 

 source, precipitate lines for common antigen- 

 antibody systems will fuse. 



Tests for the presence of the HM factor were 

 made in the manner illustrated in figure 1. Sera 

 from known HM-positive females were placed in 

 positions 1 and 4, thus placing each unknown serum 

 adjacent to an HM-positive individual. Distinct 

 positive reactions are seen in positions 5 and 0; a 

 weak positive reaction in position 2, and no reaction 

 in [losition 3. This arrangement was particularly 

 useful for positive individuals with low concentra- 

 tions of HM antigen. Although a distinct line was 

 not necessarily formed, a licnding of the control 

 line towards the unknown position, as in position 2, 

 indicated the presence of the HM factor and allowed 

 a highly sensitive test for the HM factor's presence. 



Relative concentrations of the HM factor were 

 determined by a single-diffusion method described 

 by Hayward and Augustin (1957). In this method 

 the antiserum is incorporated into the agar at 5 

 percent concentration and serial dilutions of the 

 fluid bearing the HM factor are introduced into 

 wells in the agar. The end point, the highest dilu- 

 tion at which a visible ring can be observed around 

 the well, is referred to as the titer of the solution 

 for the HM factor. Figure 2 illustrates the reactions 

 of serial dilutions of HM factor from an extract 

 from eggs of starry flounder {I'latichtijs ddlalus) 

 with the antiserum used in this study. (The pre- 

 paration of both the extract and the antiserum is 

 described below.) The end i)oiiit is at the 1/100 

 dilution. 



•The medium in this study had the followinK romposition: Difeo agar. 

 I..'i iH-reent; sociiiiiii rliloride, 0.72 percent: sodium eitrato. 0.6 percent: 

 merthiolatv, 001 percent; trypan blue. 0.01 percent. The pH was 

 adjuatt^d to 6.7, 



48 



U.S. FISH AND WILDLIFE SEUVICE 



