15.2. 1975
Speeialia
b e t w e e n 9 and 11. A t i t e r of 6 was observed w h e n antivesicle serum was tested against ganglion. H o w e v e r , the anti-ganglion serum did n o t produce positive reactions w i t h s y n a p t i c vesicles. These results would i m p l y t h a t the central nervous system shares some antigens w i t h the peripheral nervous system ~, ~4. The i m m u n o f l u o r e s c e n c e analysis revealed different histological lacolizations of antibodies from anti-ganglion and anti-vesicle sera. N a m e l y , t h e anti-ganglion serum reacted almost exclusively w i t h cell m e m b r a n e s and t h e t h i c k n e t w o r k of nerve fibres of the ganglion (FigureA), whereas the anti-vesicle serum c o m b i n e d preterentially w i t h antigens s i t u a t e d w i t h i n t h e neuron (Figure ]3). Large, round to oval nuclei, Usually excentrically placed, r e m a i n e d unstained in all e x a m i n e d sections. The staining of ganglion sections was inhibited b y absorption of antisera w i t h corresponding antigens, t h u s p o i n t i n g to t h e presence of organ-specific antigens in t h e p r e p a r a t i o n s used for i m m u n i z a t i o n . No staining was observed in control tests. A n u m b e r of ganglion sections t r e a t e d w i t h anti-vesicle serum e x h i b i t e d a faint fluorescence of inter-
15 E. BOCK, O. S. JORGENSEN and S. J. MORRIS, J. Neurochem. 22,
1013 (1974). 16 S. L. TWOMEYand F. E. SAMSONJR., Brain Res. 37, 101 (1972). IT E. DE ROBERTIS,E. G. LAPETINAand F. WALD, ExpI. Neurol. 21, 322 (1968). 16 B. W. IVIooREand D. McGREOOR,J. biol. Chem. 240, 1647 (1965). 19 G. S. BENNETTand G. M. ED~LMAN,J. biol. Chem. 243, 6234 (1968). 20 A. BIGI~AMIand D. DAHL, Brain Res. 49, 393 (1973). st This work was supported by grants from the Republic Fund for Research of Serbia, Belgrade.
241
neuronal particulates o t h e r t h a n blood vessels. This was p r o b a b l y due to the c o n t a m i n a t i o n of the s y n a p t i c vesicle p r e p a r a t i o n w i t h o t h e r antigens4,15. Besides, antibodies o t h e r t h a n those reacting w i t h s y n a p t i c vesicles, e.g. a n t i - t u b u l i n antibodies 1~, m i g h t be contributing to the staining. This aspect of cross-reactivity b e t w e e n different neuronal c o m p o n e n t s is now u n d e r investigation. R e l e v a n t to the p r o b l e m of a n t i b o d y specificity is the o b s e r v a t i o n t h a t a r a b b i t a n t i - n e r v e ending m e m b r a n e serum affects t h e a x o p l a s m and s y n a p t i c vesicles~L The present e x p e r i m e n t does not establish evidence w i t h respect to the antigenic Similarity between brain s y n a p t i c vesicles and cervical ganglion antigens, and a v a r i e t y of rat brain specific antigens ~s-20. I n s u m m i n g up, the i m m u n o f l u o r e s c e n c e analysis described here provides some basic i n f o r m a t i o n a b o u t the localizing properties of antibodies from anti-cervical ganglion and anti-brain synaptic vesicle sera, and thus justifies t h e use of those i m m u n e reagents in a functional s t u d y of the rat superior cervical ganglion~L
Rdsumd. On a d6montr6 que les anticorps contre les v6sicules s y n a p t i q u e s du cerveau du r a t se fixent de pr6f6rence duns le c y t o p l a s m e des neurones, tandis que les anticorps contre les ganglions s y m p a t h i q u e s c e r v i c a u x sup6rieurs r6agissent p r e s q u ' e x c l u s i v e m e n t avec les m e m b r a n e s des neurones du ganglion. B. D. JANKOVId, KOSANA MITROVId, J. HORVAT a n d V. SAVld
Immunology Research Center, Vojvode Stepe 458, Y - I I O 0 0 Belgrade (Yugoslavia), 6 September 1974.
Ovarian Cyst Fluid Specific Antigens The presence of o v a r i a n cyst specific antigens h a v e been d e t e c t e d earlier 1,3. A p r e l i m i n a r y report of our preparat i o n and p a r t i a l characterization of the antigens h a v e shown t h a t t h e y are glycoprotein containing 12.3% fucose ( F R G P ) 2. F u r t h e r m o r e , a n t i - F R G P a n t i b o d y reacted w i t h an e x t r a c t of h u m a n spleen. Several reports h a v e presented evidence of antigens w i t h organ specificity for h u m a n heartS,4, liver~, and b a s e m e n t membrane6, 7. R e n a l glomerular b a s e m e n t m e m b r a n e glycoproteins shared t h e i r antigenecities w i t h b a s e m e n t - m e m b r a n e - r i c h organs such as lung, p l a c e n t a and u r i n a r y glycoproteinT. L i m i t e d studies of o v a r i a n cyst c o n t e n t Were p e r f o r m e d using i m m u n o l o g i c a l procedures. The present c o m m u n i cation describes the chemical and immunological properties of F R G P of o v a r i a n cyst origin. Materials and methods. A p p r o x i m a t e l y 200 1 of ovarian cyst fluid f r o m a p a t i e n t w i t h o v a r i a n c y s t o m a were collected and filtered. A f t e r 10 1 of the fluid were m i x e d w i t h 3.62 kg of solid a m m o n i u m sulfate and 30 ml of c o n c e n t r a t e d hydrochloric acid to p H 4.3, the m i x t u r e was allowed to stabilize for 20 h at 4~ The centrifuged supernate was acidified w i t h c o n c e n t r a t e d hydrochloric acid to p H 3.7, and t h e n 2.31 kg of a m m o n i u m sulfate were added for 100% saturation. A f t e r a 20 h period at 4~ the p r e c i p i t a t e d crude glycoprotein (CGP) was r e c o v e r e d b y centrifugation, dialyzed and lyophilized. E t h a n o l was added to a 2% solution of CGP in stepwise fashion to give concentrations of 38%, 66% and 80% (v/v) at 4~ A glycoprotein fraction o b t a i n e d at 38-
66% (v/v) ethanol c o n c e n t r a t i o n (first stage) was refractionated using t h e ethanol procedure described above. The precipitate formed at 38-66 % (v/v) ethanol concentrations was centrifuged at 24,000 g for 80 min at 4~ Tile s u p e r n a t a n t (1~R G P ) was lyophilized. Frozen p o s t - m o r t e m tissues from p u l m o n a r y c a r c i n o m a p a t i e n t s were t h a w e d and homogenized in cold distilled water. Soluble constituents were collected b y centrifugation and lyophilized. A p p r o x i m a t e l y 100 m g of t h e distilled w a t e r e x t r a c t were dissolved in 30 mt of distilled w a t e r and subjected to 'first stage' ethanol fractionation. Pseudomucins were a gift of Dr. Y. NAGAI, F u k u s h i m a Medical College. R a b b i t antisera against F R G P were p r e p a r e d b y r e p e a t e d injections o f t h e antigen w i t h F r e u n d ' s c o m p l e t e a d j u v a n t (Ditto). The antisera were absorbed w i t h lyophilized pooled n o r m a l h u m a n serum s. I m m u n o l o g i c a l analyses
1 L. ODIN, Acta Soc. Med. upsal. 64, 25 (1959). M. HORIKAWA, M. KUROKAWAand K. HOTTA, Seikagaku 42, 415 (1970). 8 E. ESPINOSA and M. H. KAPLA~, J. Immun. lOG 1020 (1968). 4 ]~. I~SPINOSAand M. H. KAPLAN~ J. Immun. 705, 416 (1970). 5 K. H. IVIEYERZUM BOSCHENFELD and P. A. MIESCHER, Clin. exp.
Immun. 70, 89 (1972). 6 j. j. MCPHAULJR. and F. J. DIXON, J. exp. Med. 730, 1395
(1969). K. IV[.HAWKINGand F. ~V[ILGROM,Int. Arch. Allergy d3, 641 (1972). 8 p. BURTIN, S. VON~IKLEISTarid D. BIJFFE, Bull. Soc. Chim. biol. dg, 1389 (1967).
Specialia
242
EXPERIENTIA
31/2
Yield and chemical eompositiou of glyeoproteins Fraetionation stage
Ammonium sulfate fractionation (crude glycoprotein) Ethanol fraetionation (fucose-rieh glycoprotein)
Yield {% of amount present in the original ovarian cyst content)
Chemical composition (~o) ~ Fucose
N-aeetyl neuraminic acid
Galactose
Hexosamine ~
Protein
21.3
5.3
1.6
9.3
12.0
63.0
0.7
12.3
2.8
17.2
16.4
58.5
Values expressed as dry weight basis, b Determined after hydrolysis of samples in 2 N HC1 for 4 h at 100~ 16.
were carried o u t u s i n g t h e O u c h t e r l o n y d o u b l e diffusion p r o c e d u r e 9 a n d also t h e i m m u n o e l e c t r o p h o r e t i c m e t h o d of SCHEIDEGGI~R 1~ M e a s u r e m e n t of hexose, fucose, a n d sialic acid c o n s t i t u e n t s was a c c o m p l i s h e d b y m e a n s of t h e orcinollt, GIBBONSIS a n d WARRENIS procedures, respectively. T h e m e t h o d of LOWRY et al.! ~ was used for p r o t e i n analysis, w i t h b o v i n e s e r u m a l b u m i n as s t a n d a r d . , Results and discussion. Recovery and composition data for t h e 2 g l y c o p r o t e i n f r a c t i o n s are s h o w n in t h e Table. T h e i n i t i a l yield of F R G P was 0.61 g f r o m 10 l of o v a r i a n c y s t fluid. G a l a c t o s e a n d fucose were i d e n t i f i e d b y gas liquid c h r o m a t o g r a p h y is as t h e only n e u t r a l s u g a r c o m p o n e n t s p r e s e n t ; m a n n o s e was n o t d e t e c t e d . Glucosa m i n e a n d g a l a c t o s a m i n e were d e t e c t e d ~5, ~6. T h e t o t a l
Fig. 1. Ouchtertony results showing the antigenic relationship of fueose-rich glycoprotein (FRGP), 4-Sp, 8-Sp and kidney (7-K). Central well contains anti-fucose-rich glycoprotein serum absorbed with normal human serum.
c a r b o h y d r a t e c o n t e n t of t h e C G P was 28.2~o w h i l e t h a t of F R G P was 48.7%. R e s u l t s of t h e c a r b a z o l e - s u l f u r i c acid t e s t 17 were n e g a t i v e . T h e a n t i - F R G P s e r u m a b s o r b e d w i t h n o r m a l h u m a n s e r u m ( A b - N H S ) used i n t h i s w o r k d i d n o t p r o d u c e p r e c i t i n lines w h e n t e s t e d w i t h pooled n o r m a l s e r u m or s e r u m o b t a i n e d f r o m t h e p a t i e n t w h o s e o v a r i a n c y s t fluid was used in t h i s s t u d y . I m m u n o e l e c t r o p h o r e s i s of F R G P a g a i n s t a n t i s e r u m p r o d u c e d 4 or 5 b r o a d a n d less s y m m e t r i c a l p r e c i p i t i n lines, in t h e e~- to y-globulin region. 5 p r e c i p i t i n lines were elicited u s i n g t h e O u c h t e r l o n y m e t h o d . T h r e e of 711 p a t h o l o g i c a l sera p r o v e d to h a v e a n a n t i g e n i c s u b s t a n c e w h i c h r e a c t e d w i t h A b - N H S . No c r o s s - r e a c t i o n of t h e a n t i s e r u m w i t h 28 ascitic a n d 2 p l e u r a l fluids w a s o b s e r v e d . P o s t - m o r t e m tissue e x t r a c t s , of liver, k i d n e y , spleen, large i n t e s t i n e , a b d o m i n a l muscle, g r e a t a m e n t n m , a n d l u n g were t e s t e d w i t h A b - N H S b y t h e O u c h t e r l o n y p r o c e d u r e . Of these, 2 spleen s a m p l e s (4-Sp a n d 8-Sp) r e a c t e d w i t h t h e a n t i s e r u m to f o r m a p r e c i p i t i n line of c o m p l e t e i d e n t i t y w i t h e a c h o t h e r a n d s h o w e d i m m u n o l o g i c a l i d e n t i t y wittl one of t h e p r e c i p i t i n lines f o r m e d b y F R G P a g a i n s t t h e a n t i s e r u m (Figure 1). 4 p s e u d o m u c i n e s were t e s t e d , a n d one of t h e m was f o u n d to r e a c t w i t h t h e a n t i s e r u m b y f o r m i n g 2 p r e c i p i t i n lines. W h e n t h e a n t i s e r u m was a b s o r b e d w i t h t h e spleen s a m p l e (4-Sp), t h e a b s o r b e d a n t i b o d y failed to f o r m t h e p r e c i p i t i n line a g a i n s t 4-Sp a n d 8-Sp (Figure 2) ; h o w e v e r , p r e c i p i t i n lines still f o r m e d b e t w e e n pseudom u c i n a n d t h e a n t i b o d y w i t h 4-Sp. T h e p r e c e d i n g d a t a i n d i c a t e t h e p r e s e n c e of a t l e a s t 5 a n t i g e n s in F R G P . Of t h e 5 a n t i g e n s , 2 a n t i g e n s are c o m m o n to p s e u d o m u c i n e (1 o u t of 4 tested) a n d 1 a n t i g e n is a t t r i b u t a b l e t o spleen. A d d i t i o n a l a n t i g e n s e x h i b i t t h e o v a r i a n cyst c o n t e n t speccificity. F R G P e x h i b i t e d b o t h A a n d H b l o o d a c t i v i t i e s by the hem agglutination inhibition technique. Our study s h o w e d t h a t t h e c a r b o h y d r a t e c o m p o s i t i o n of F1RGP is
9 0. OUCHTERLONY, Progr. Allergy 5, 1 (1958); 6, 30 (1962). 10 3. j. SCHEIDEGGER, Int. Arch. Allergy 7, 103 (1955). 11 R. J. WINZLER, Method of Biochemical Analysis (Ed. D. GLIC~r Interseience, New York 1955), voI. 2, p. 279. 12 M. N. GIBBONS,Analyst 80, 268 (1955). 18 L. WARREN, J. biol. Chem. 234, 1971 (1959). 14 O. H. LOWRY, N. J. ROSEBROIJGH, A. L. •ARR and R. J. RANDALL, J. biol. Chem. 193, 256 (1951). 15 T. YAMAKAWA, N. U E T A and I. ISHIZUKA, Jap. J. exp. Med. 34, 231
Fig. 2. 0.I ml of the anti-fucose-rieh glycoprotein serum absorbed with normal human serum (Ab-NHS) was absorbed with 1 mg of 4-Sp. The absorbed antibody (Ab-NHS-4-Sp) failed to form any precipitin line against the 4-Sp.
(1964). 16 1~. BLEBHER, A. J. NUTTER and C. M. SAMBROOK,Analyst 79, 201
(1954). IV Z. DlSCHE, J. biol. Chenl. 767, 189 (1947).
15.2. 1975
243
Specialia
d i f f e r e n t f r o m F R G P s in urine a n d p a r o t i d e saliva ls-~~ S u b s t a n c e s of t h i s t y p e f o u n d in o v a r i a n c y s t m a t e r i a l s e e m t o b e a k i n d of b l o o d - g r o u p s u b s t a n c e t y p e ~x. I t h a s
Glycoproteins (Ed. A. GOTTSHALK; Elsevier Publishing Company, Amsterdam 1972), part B, p. 909. 19 R. D. FRIEDMAN, A. D. ]VfERR1TT and D. BIXLER, Bioehim. biophys. Acta 230, 599 (1971). 2o M. J. LEVINE, S. A. ELLISOIgand O. P. BAHL,Archs oral Biol. 78, 827 (1973). 21 W. M. WATKINS; Glycoporteins (Ed. A. GOTTSHALK; Elsevier Publishing Company, Amsterdam 1972), part B, p. 830. 22 School of Hygienic Sciences, Kitasato University. 2z Department. of Biochemistry, School of Medicine, Kitasato University, to whom correspondence should be directed. 18 R. BOURR1LLON,
been shown that this F R G P from ovarian cyst content shares a c o m m o n a n t i g e n e c i t y w i t h spleen tissue.
Zusammen/assung. F u c o s e - r e i c h e s G l y k o p r o t e i n w u r d e aus E i e r s t o c k p s e u d o m u c i n isoliert. Mit Hilfe spezifischer A n t i - G l y k o p r o t e i n - S e r e n w u r d e die A n t i g e n i z i t ~ t der P s e u d o m u c i n e m i t I m m u n o e l e k t r o p h o r e s e u n d die Ouchterlony Methode untersucht. M. g . HAMAZAK132 a n d K. HOT'rA~S
School o[ Hygienic Sciences, and Department o/Biochemistry, School o[ Medicine Kitasato University Asamizodai, Sagamihara, Kanagawa (Japan), 12 July 7974. :
if IL.
Effect of R e m o v i n g o n e O v a r y and a Half on O v u l a t i o n N u m b e r in C y c l i n g R a t s The L a w of follicular c o n s t a n c y i n d i c a t e s t h a t a g i v e n n u m b e r of follicles m a t u r e d u r i n g each e s t r o u s cycle regardless of t h e a m o u n t of o v a r i a n tissue p r e s e n t . W h e t h e r t h i s s a m e g e n e r a l i z a t i o n applies t o t h e n u m b e r of o v a shed, r e m a i n s t o be a n s w e r e d . If it does, t h e n t h e o v u l a t i o n n u m b e r for a n a n i m a l should n o t c h a n g e w h e n o v a r i a n tissue is r e m o v e d or w h e n e x t r a - o v a r i a n tissue is a d d e d . I n a g r e e m e n t is t h e f a c t t h a t u n i l a t e r M o v a r i e c t o m y in i n t a c t 4- a n d 5-day cycling r a t s r e s u l t e d in t h e r e m a i n i n g o v a r y d o u b l i n g t h e eggs s h e d 1. H o w e v e r , conflicting w i t h t h i s idea is t h e f i n d i n g t h a t t w o e x t r a - o v a r i a n allografts in F i s c h e r 344 r a t s caused a d e c r e a s e in t h e n u m b e r of eggs o v u l a t e d f r o m t h e in situ ovaries (CmHAL a n d PEPPLER, u n p u b l i s h e d ) . A t 7-8 weeks p o s t - o p e r a t i v e in h e m i c a s t r a t e d r a t s or t h o s e in w h i c h one o v a r y a n d a half h a d b e e n r e m o v e d , t h e r e m a i n i n g o v a r i a n tissue c o n t a i n e d t h e s a m e n u m b e r of large follicles 2. H o w e v e r , h y p e r t r o p h i e d half ovaries left in situ only c o n t a i n e d 6 fresh c o r p o r a l u t e s 5 weeks a f t e r t h e r e m o v a l of one o v a r y a n d a half 3. B e c a u s e of t h e implied discrepancy between these two reports, this i n v e s t i g a t i o n was p e r f o r m e d t o d e t e r m i n e if r e m o v i n g one o v a r y a n d a half for 1 e s t r o u s cycle r e s u l t e d in t h e n o r m a l n u m b e r of eggs b e i n g o v u l a t e d ( c o m p e n s a t o r y ovulation) a t t h e n e x t estrus. t-Ioltzman, female virgin r a t s were received a t 50 d a y s of age a n d d i v i d e d i n t o c o n t r o l (N = 8) a n d e x p e r i m e n t a l (N = 10) groups. T h e a n i m a l s were m a i n t a i n e d in g r o u p s
of 2 p e r cage w i t h w a t e r a n d l a b o r a t o r y c h o w p r o v i d e d ad l i b i t u m . T h e l i g h t i n g schedule w a s r e g u l a t e d for 14 h o~ i l l u m i n a t i o n a n d 10 h of darkness. Daily v a g i n a l s m e a r s were t a k e n u n t i l 3 cycles were observed. D a y 1 of t h e cycle refers t o estrus. S e v e n t y - f i v e p e r c e n t of t h e o v a r i a n tissue was r e m o v e d on d a y 2 of t h e cycle b y a d o r s a l - l a t e r a l a p p r o a c h . One o v a r y (18.5 • 1.7 mg/100 g b o d y weight) w a s c o m p l e t e l y r e m o v e d a n d half (7.9 • 1.5 mg/100 g b o d y weight) of t h e o t h e r o v a r y was r e m o v e d e i t h e r b y m a k i n g a t r a n s v e r s e cut across b o t h t h e s u p e r i o r a n d inferior ends of t h e o v a r y or b y m a k i n g a lateral p a r a s a g i t t a l cut. T h e e n d s of t h e o v a r i a n b u r s a were pulled a r o u n d t h e r e m a i n i n g o v a r i a n fragment and pressed together. A n i m a l s were killed 1 v a g i n a l cycle l a t e r on d a y 2 (metestrus). B o d y w e i g h t a n d v a r i o u s o r g a n w e i g h t s were recorded. Each oviduct was dissected from the ovary (control group) or o v a r i a n f r a g m e n t ( e x p e r i m e n t a l group) a n d f l u s h e d w i t h n o r m a l saline to d e t e r m i n e o v u l a t i o n number.
t R. D. PEFPLER and G. S. GREENWALD, Am. J. Anat. /27, 1 (1970). A. M. MAMMY,S. ZUCKERMANand H. D. PATTERSON, J. Endoer. 8, 347 (1952). L. B. KWAKYE, A. RHIMtgR and J. VAJDA, Acts morph, neerl.scand. 9, 303 (1971/1972).
Effect of removing one ovary and a half on ovulation number and various organ weights Group
Control (N = s) One ovary and a half removed (N = ~0/
Body weight (g) :[: S.E.
Eggs ovulated per rat =[: S.E.
Organ weights (rag/100 g body wt. ~c S.E. Ovary ~
Uterus
Adrenalb
216.7 =L 2.8
9.5 • 0.6
16.1 -t- 0.8
117.5 -4- 2.8
26.1 4- 1.2
212.3 4-2.8
5.1 :t_ 0.6 c
18.8 :Jzl.4
114.3+ 4.7
30.2 zt= 1.0 o
a Mean ovarian weight is one-half of the sum of both ovaries for control rats and the weight of the remaining ovarian fi'agment for the experimental group, b Mean adrenal weight is both adrenals for all rats. o P < 0.025 when compared to value for control rats.
Speeialia
b e t w e e n 9 and 11. A t i t e r of 6 was observed w h e n antivesicle serum was tested against ganglion. H o w e v e r , the anti-ganglion serum did n o t produce positive reactions w i t h s y n a p t i c vesicles. These results would i m p l y t h a t the central nervous system shares some antigens w i t h the peripheral nervous system ~, ~4. The i m m u n o f l u o r e s c e n c e analysis revealed different histological lacolizations of antibodies from anti-ganglion and anti-vesicle sera. N a m e l y , t h e anti-ganglion serum reacted almost exclusively w i t h cell m e m b r a n e s and t h e t h i c k n e t w o r k of nerve fibres of the ganglion (FigureA), whereas the anti-vesicle serum c o m b i n e d preterentially w i t h antigens s i t u a t e d w i t h i n t h e neuron (Figure ]3). Large, round to oval nuclei, Usually excentrically placed, r e m a i n e d unstained in all e x a m i n e d sections. The staining of ganglion sections was inhibited b y absorption of antisera w i t h corresponding antigens, t h u s p o i n t i n g to t h e presence of organ-specific antigens in t h e p r e p a r a t i o n s used for i m m u n i z a t i o n . No staining was observed in control tests. A n u m b e r of ganglion sections t r e a t e d w i t h anti-vesicle serum e x h i b i t e d a faint fluorescence of inter-
15 E. BOCK, O. S. JORGENSEN and S. J. MORRIS, J. Neurochem. 22,
1013 (1974). 16 S. L. TWOMEYand F. E. SAMSONJR., Brain Res. 37, 101 (1972). IT E. DE ROBERTIS,E. G. LAPETINAand F. WALD, ExpI. Neurol. 21, 322 (1968). 16 B. W. IVIooREand D. McGREOOR,J. biol. Chem. 240, 1647 (1965). 19 G. S. BENNETTand G. M. ED~LMAN,J. biol. Chem. 243, 6234 (1968). 20 A. BIGI~AMIand D. DAHL, Brain Res. 49, 393 (1973). st This work was supported by grants from the Republic Fund for Research of Serbia, Belgrade.
241
neuronal particulates o t h e r t h a n blood vessels. This was p r o b a b l y due to the c o n t a m i n a t i o n of the s y n a p t i c vesicle p r e p a r a t i o n w i t h o t h e r antigens4,15. Besides, antibodies o t h e r t h a n those reacting w i t h s y n a p t i c vesicles, e.g. a n t i - t u b u l i n antibodies 1~, m i g h t be contributing to the staining. This aspect of cross-reactivity b e t w e e n different neuronal c o m p o n e n t s is now u n d e r investigation. R e l e v a n t to the p r o b l e m of a n t i b o d y specificity is the o b s e r v a t i o n t h a t a r a b b i t a n t i - n e r v e ending m e m b r a n e serum affects t h e a x o p l a s m and s y n a p t i c vesicles~L The present e x p e r i m e n t does not establish evidence w i t h respect to the antigenic Similarity between brain s y n a p t i c vesicles and cervical ganglion antigens, and a v a r i e t y of rat brain specific antigens ~s-20. I n s u m m i n g up, the i m m u n o f l u o r e s c e n c e analysis described here provides some basic i n f o r m a t i o n a b o u t the localizing properties of antibodies from anti-cervical ganglion and anti-brain synaptic vesicle sera, and thus justifies t h e use of those i m m u n e reagents in a functional s t u d y of the rat superior cervical ganglion~L
Rdsumd. On a d6montr6 que les anticorps contre les v6sicules s y n a p t i q u e s du cerveau du r a t se fixent de pr6f6rence duns le c y t o p l a s m e des neurones, tandis que les anticorps contre les ganglions s y m p a t h i q u e s c e r v i c a u x sup6rieurs r6agissent p r e s q u ' e x c l u s i v e m e n t avec les m e m b r a n e s des neurones du ganglion. B. D. JANKOVId, KOSANA MITROVId, J. HORVAT a n d V. SAVld
Immunology Research Center, Vojvode Stepe 458, Y - I I O 0 0 Belgrade (Yugoslavia), 6 September 1974.
Ovarian Cyst Fluid Specific Antigens The presence of o v a r i a n cyst specific antigens h a v e been d e t e c t e d earlier 1,3. A p r e l i m i n a r y report of our preparat i o n and p a r t i a l characterization of the antigens h a v e shown t h a t t h e y are glycoprotein containing 12.3% fucose ( F R G P ) 2. F u r t h e r m o r e , a n t i - F R G P a n t i b o d y reacted w i t h an e x t r a c t of h u m a n spleen. Several reports h a v e presented evidence of antigens w i t h organ specificity for h u m a n heartS,4, liver~, and b a s e m e n t membrane6, 7. R e n a l glomerular b a s e m e n t m e m b r a n e glycoproteins shared t h e i r antigenecities w i t h b a s e m e n t - m e m b r a n e - r i c h organs such as lung, p l a c e n t a and u r i n a r y glycoproteinT. L i m i t e d studies of o v a r i a n cyst c o n t e n t Were p e r f o r m e d using i m m u n o l o g i c a l procedures. The present c o m m u n i cation describes the chemical and immunological properties of F R G P of o v a r i a n cyst origin. Materials and methods. A p p r o x i m a t e l y 200 1 of ovarian cyst fluid f r o m a p a t i e n t w i t h o v a r i a n c y s t o m a were collected and filtered. A f t e r 10 1 of the fluid were m i x e d w i t h 3.62 kg of solid a m m o n i u m sulfate and 30 ml of c o n c e n t r a t e d hydrochloric acid to p H 4.3, the m i x t u r e was allowed to stabilize for 20 h at 4~ The centrifuged supernate was acidified w i t h c o n c e n t r a t e d hydrochloric acid to p H 3.7, and t h e n 2.31 kg of a m m o n i u m sulfate were added for 100% saturation. A f t e r a 20 h period at 4~ the p r e c i p i t a t e d crude glycoprotein (CGP) was r e c o v e r e d b y centrifugation, dialyzed and lyophilized. E t h a n o l was added to a 2% solution of CGP in stepwise fashion to give concentrations of 38%, 66% and 80% (v/v) at 4~ A glycoprotein fraction o b t a i n e d at 38-
66% (v/v) ethanol c o n c e n t r a t i o n (first stage) was refractionated using t h e ethanol procedure described above. The precipitate formed at 38-66 % (v/v) ethanol concentrations was centrifuged at 24,000 g for 80 min at 4~ Tile s u p e r n a t a n t (1~R G P ) was lyophilized. Frozen p o s t - m o r t e m tissues from p u l m o n a r y c a r c i n o m a p a t i e n t s were t h a w e d and homogenized in cold distilled water. Soluble constituents were collected b y centrifugation and lyophilized. A p p r o x i m a t e l y 100 m g of t h e distilled w a t e r e x t r a c t were dissolved in 30 mt of distilled w a t e r and subjected to 'first stage' ethanol fractionation. Pseudomucins were a gift of Dr. Y. NAGAI, F u k u s h i m a Medical College. R a b b i t antisera against F R G P were p r e p a r e d b y r e p e a t e d injections o f t h e antigen w i t h F r e u n d ' s c o m p l e t e a d j u v a n t (Ditto). The antisera were absorbed w i t h lyophilized pooled n o r m a l h u m a n serum s. I m m u n o l o g i c a l analyses
1 L. ODIN, Acta Soc. Med. upsal. 64, 25 (1959). M. HORIKAWA, M. KUROKAWAand K. HOTTA, Seikagaku 42, 415 (1970). 8 E. ESPINOSA and M. H. KAPLA~, J. Immun. lOG 1020 (1968). 4 ]~. I~SPINOSAand M. H. KAPLAN~ J. Immun. 705, 416 (1970). 5 K. H. IVIEYERZUM BOSCHENFELD and P. A. MIESCHER, Clin. exp.
Immun. 70, 89 (1972). 6 j. j. MCPHAULJR. and F. J. DIXON, J. exp. Med. 730, 1395
(1969). K. IV[.HAWKINGand F. ~V[ILGROM,Int. Arch. Allergy d3, 641 (1972). 8 p. BURTIN, S. VON~IKLEISTarid D. BIJFFE, Bull. Soc. Chim. biol. dg, 1389 (1967).
Specialia
242
EXPERIENTIA
31/2
Yield and chemical eompositiou of glyeoproteins Fraetionation stage
Ammonium sulfate fractionation (crude glycoprotein) Ethanol fraetionation (fucose-rieh glycoprotein)
Yield {% of amount present in the original ovarian cyst content)
Chemical composition (~o) ~ Fucose
N-aeetyl neuraminic acid
Galactose
Hexosamine ~
Protein
21.3
5.3
1.6
9.3
12.0
63.0
0.7
12.3
2.8
17.2
16.4
58.5
Values expressed as dry weight basis, b Determined after hydrolysis of samples in 2 N HC1 for 4 h at 100~ 16.
were carried o u t u s i n g t h e O u c h t e r l o n y d o u b l e diffusion p r o c e d u r e 9 a n d also t h e i m m u n o e l e c t r o p h o r e t i c m e t h o d of SCHEIDEGGI~R 1~ M e a s u r e m e n t of hexose, fucose, a n d sialic acid c o n s t i t u e n t s was a c c o m p l i s h e d b y m e a n s of t h e orcinollt, GIBBONSIS a n d WARRENIS procedures, respectively. T h e m e t h o d of LOWRY et al.! ~ was used for p r o t e i n analysis, w i t h b o v i n e s e r u m a l b u m i n as s t a n d a r d . , Results and discussion. Recovery and composition data for t h e 2 g l y c o p r o t e i n f r a c t i o n s are s h o w n in t h e Table. T h e i n i t i a l yield of F R G P was 0.61 g f r o m 10 l of o v a r i a n c y s t fluid. G a l a c t o s e a n d fucose were i d e n t i f i e d b y gas liquid c h r o m a t o g r a p h y is as t h e only n e u t r a l s u g a r c o m p o n e n t s p r e s e n t ; m a n n o s e was n o t d e t e c t e d . Glucosa m i n e a n d g a l a c t o s a m i n e were d e t e c t e d ~5, ~6. T h e t o t a l
Fig. 1. Ouchtertony results showing the antigenic relationship of fueose-rich glycoprotein (FRGP), 4-Sp, 8-Sp and kidney (7-K). Central well contains anti-fucose-rich glycoprotein serum absorbed with normal human serum.
c a r b o h y d r a t e c o n t e n t of t h e C G P was 28.2~o w h i l e t h a t of F R G P was 48.7%. R e s u l t s of t h e c a r b a z o l e - s u l f u r i c acid t e s t 17 were n e g a t i v e . T h e a n t i - F R G P s e r u m a b s o r b e d w i t h n o r m a l h u m a n s e r u m ( A b - N H S ) used i n t h i s w o r k d i d n o t p r o d u c e p r e c i t i n lines w h e n t e s t e d w i t h pooled n o r m a l s e r u m or s e r u m o b t a i n e d f r o m t h e p a t i e n t w h o s e o v a r i a n c y s t fluid was used in t h i s s t u d y . I m m u n o e l e c t r o p h o r e s i s of F R G P a g a i n s t a n t i s e r u m p r o d u c e d 4 or 5 b r o a d a n d less s y m m e t r i c a l p r e c i p i t i n lines, in t h e e~- to y-globulin region. 5 p r e c i p i t i n lines were elicited u s i n g t h e O u c h t e r l o n y m e t h o d . T h r e e of 711 p a t h o l o g i c a l sera p r o v e d to h a v e a n a n t i g e n i c s u b s t a n c e w h i c h r e a c t e d w i t h A b - N H S . No c r o s s - r e a c t i o n of t h e a n t i s e r u m w i t h 28 ascitic a n d 2 p l e u r a l fluids w a s o b s e r v e d . P o s t - m o r t e m tissue e x t r a c t s , of liver, k i d n e y , spleen, large i n t e s t i n e , a b d o m i n a l muscle, g r e a t a m e n t n m , a n d l u n g were t e s t e d w i t h A b - N H S b y t h e O u c h t e r l o n y p r o c e d u r e . Of these, 2 spleen s a m p l e s (4-Sp a n d 8-Sp) r e a c t e d w i t h t h e a n t i s e r u m to f o r m a p r e c i p i t i n line of c o m p l e t e i d e n t i t y w i t h e a c h o t h e r a n d s h o w e d i m m u n o l o g i c a l i d e n t i t y wittl one of t h e p r e c i p i t i n lines f o r m e d b y F R G P a g a i n s t t h e a n t i s e r u m (Figure 1). 4 p s e u d o m u c i n e s were t e s t e d , a n d one of t h e m was f o u n d to r e a c t w i t h t h e a n t i s e r u m b y f o r m i n g 2 p r e c i p i t i n lines. W h e n t h e a n t i s e r u m was a b s o r b e d w i t h t h e spleen s a m p l e (4-Sp), t h e a b s o r b e d a n t i b o d y failed to f o r m t h e p r e c i p i t i n line a g a i n s t 4-Sp a n d 8-Sp (Figure 2) ; h o w e v e r , p r e c i p i t i n lines still f o r m e d b e t w e e n pseudom u c i n a n d t h e a n t i b o d y w i t h 4-Sp. T h e p r e c e d i n g d a t a i n d i c a t e t h e p r e s e n c e of a t l e a s t 5 a n t i g e n s in F R G P . Of t h e 5 a n t i g e n s , 2 a n t i g e n s are c o m m o n to p s e u d o m u c i n e (1 o u t of 4 tested) a n d 1 a n t i g e n is a t t r i b u t a b l e t o spleen. A d d i t i o n a l a n t i g e n s e x h i b i t t h e o v a r i a n cyst c o n t e n t speccificity. F R G P e x h i b i t e d b o t h A a n d H b l o o d a c t i v i t i e s by the hem agglutination inhibition technique. Our study s h o w e d t h a t t h e c a r b o h y d r a t e c o m p o s i t i o n of F1RGP is
9 0. OUCHTERLONY, Progr. Allergy 5, 1 (1958); 6, 30 (1962). 10 3. j. SCHEIDEGGER, Int. Arch. Allergy 7, 103 (1955). 11 R. J. WINZLER, Method of Biochemical Analysis (Ed. D. GLIC~r Interseience, New York 1955), voI. 2, p. 279. 12 M. N. GIBBONS,Analyst 80, 268 (1955). 18 L. WARREN, J. biol. Chem. 234, 1971 (1959). 14 O. H. LOWRY, N. J. ROSEBROIJGH, A. L. •ARR and R. J. RANDALL, J. biol. Chem. 193, 256 (1951). 15 T. YAMAKAWA, N. U E T A and I. ISHIZUKA, Jap. J. exp. Med. 34, 231
Fig. 2. 0.I ml of the anti-fucose-rieh glycoprotein serum absorbed with normal human serum (Ab-NHS) was absorbed with 1 mg of 4-Sp. The absorbed antibody (Ab-NHS-4-Sp) failed to form any precipitin line against the 4-Sp.
(1964). 16 1~. BLEBHER, A. J. NUTTER and C. M. SAMBROOK,Analyst 79, 201
(1954). IV Z. DlSCHE, J. biol. Chenl. 767, 189 (1947).
15.2. 1975
243
Specialia
d i f f e r e n t f r o m F R G P s in urine a n d p a r o t i d e saliva ls-~~ S u b s t a n c e s of t h i s t y p e f o u n d in o v a r i a n c y s t m a t e r i a l s e e m t o b e a k i n d of b l o o d - g r o u p s u b s t a n c e t y p e ~x. I t h a s
Glycoproteins (Ed. A. GOTTSHALK; Elsevier Publishing Company, Amsterdam 1972), part B, p. 909. 19 R. D. FRIEDMAN, A. D. ]VfERR1TT and D. BIXLER, Bioehim. biophys. Acta 230, 599 (1971). 2o M. J. LEVINE, S. A. ELLISOIgand O. P. BAHL,Archs oral Biol. 78, 827 (1973). 21 W. M. WATKINS; Glycoporteins (Ed. A. GOTTSHALK; Elsevier Publishing Company, Amsterdam 1972), part B, p. 830. 22 School of Hygienic Sciences, Kitasato University. 2z Department. of Biochemistry, School of Medicine, Kitasato University, to whom correspondence should be directed. 18 R. BOURR1LLON,
been shown that this F R G P from ovarian cyst content shares a c o m m o n a n t i g e n e c i t y w i t h spleen tissue.
Zusammen/assung. F u c o s e - r e i c h e s G l y k o p r o t e i n w u r d e aus E i e r s t o c k p s e u d o m u c i n isoliert. Mit Hilfe spezifischer A n t i - G l y k o p r o t e i n - S e r e n w u r d e die A n t i g e n i z i t ~ t der P s e u d o m u c i n e m i t I m m u n o e l e k t r o p h o r e s e u n d die Ouchterlony Methode untersucht. M. g . HAMAZAK132 a n d K. HOT'rA~S
School o[ Hygienic Sciences, and Department o/Biochemistry, School o[ Medicine Kitasato University Asamizodai, Sagamihara, Kanagawa (Japan), 12 July 7974. :
if IL.
Effect of R e m o v i n g o n e O v a r y and a Half on O v u l a t i o n N u m b e r in C y c l i n g R a t s The L a w of follicular c o n s t a n c y i n d i c a t e s t h a t a g i v e n n u m b e r of follicles m a t u r e d u r i n g each e s t r o u s cycle regardless of t h e a m o u n t of o v a r i a n tissue p r e s e n t . W h e t h e r t h i s s a m e g e n e r a l i z a t i o n applies t o t h e n u m b e r of o v a shed, r e m a i n s t o be a n s w e r e d . If it does, t h e n t h e o v u l a t i o n n u m b e r for a n a n i m a l should n o t c h a n g e w h e n o v a r i a n tissue is r e m o v e d or w h e n e x t r a - o v a r i a n tissue is a d d e d . I n a g r e e m e n t is t h e f a c t t h a t u n i l a t e r M o v a r i e c t o m y in i n t a c t 4- a n d 5-day cycling r a t s r e s u l t e d in t h e r e m a i n i n g o v a r y d o u b l i n g t h e eggs s h e d 1. H o w e v e r , conflicting w i t h t h i s idea is t h e f i n d i n g t h a t t w o e x t r a - o v a r i a n allografts in F i s c h e r 344 r a t s caused a d e c r e a s e in t h e n u m b e r of eggs o v u l a t e d f r o m t h e in situ ovaries (CmHAL a n d PEPPLER, u n p u b l i s h e d ) . A t 7-8 weeks p o s t - o p e r a t i v e in h e m i c a s t r a t e d r a t s or t h o s e in w h i c h one o v a r y a n d a half h a d b e e n r e m o v e d , t h e r e m a i n i n g o v a r i a n tissue c o n t a i n e d t h e s a m e n u m b e r of large follicles 2. H o w e v e r , h y p e r t r o p h i e d half ovaries left in situ only c o n t a i n e d 6 fresh c o r p o r a l u t e s 5 weeks a f t e r t h e r e m o v a l of one o v a r y a n d a half 3. B e c a u s e of t h e implied discrepancy between these two reports, this i n v e s t i g a t i o n was p e r f o r m e d t o d e t e r m i n e if r e m o v i n g one o v a r y a n d a half for 1 e s t r o u s cycle r e s u l t e d in t h e n o r m a l n u m b e r of eggs b e i n g o v u l a t e d ( c o m p e n s a t o r y ovulation) a t t h e n e x t estrus. t-Ioltzman, female virgin r a t s were received a t 50 d a y s of age a n d d i v i d e d i n t o c o n t r o l (N = 8) a n d e x p e r i m e n t a l (N = 10) groups. T h e a n i m a l s were m a i n t a i n e d in g r o u p s
of 2 p e r cage w i t h w a t e r a n d l a b o r a t o r y c h o w p r o v i d e d ad l i b i t u m . T h e l i g h t i n g schedule w a s r e g u l a t e d for 14 h o~ i l l u m i n a t i o n a n d 10 h of darkness. Daily v a g i n a l s m e a r s were t a k e n u n t i l 3 cycles were observed. D a y 1 of t h e cycle refers t o estrus. S e v e n t y - f i v e p e r c e n t of t h e o v a r i a n tissue was r e m o v e d on d a y 2 of t h e cycle b y a d o r s a l - l a t e r a l a p p r o a c h . One o v a r y (18.5 • 1.7 mg/100 g b o d y weight) w a s c o m p l e t e l y r e m o v e d a n d half (7.9 • 1.5 mg/100 g b o d y weight) of t h e o t h e r o v a r y was r e m o v e d e i t h e r b y m a k i n g a t r a n s v e r s e cut across b o t h t h e s u p e r i o r a n d inferior ends of t h e o v a r y or b y m a k i n g a lateral p a r a s a g i t t a l cut. T h e e n d s of t h e o v a r i a n b u r s a were pulled a r o u n d t h e r e m a i n i n g o v a r i a n fragment and pressed together. A n i m a l s were killed 1 v a g i n a l cycle l a t e r on d a y 2 (metestrus). B o d y w e i g h t a n d v a r i o u s o r g a n w e i g h t s were recorded. Each oviduct was dissected from the ovary (control group) or o v a r i a n f r a g m e n t ( e x p e r i m e n t a l group) a n d f l u s h e d w i t h n o r m a l saline to d e t e r m i n e o v u l a t i o n number.
t R. D. PEFPLER and G. S. GREENWALD, Am. J. Anat. /27, 1 (1970). A. M. MAMMY,S. ZUCKERMANand H. D. PATTERSON, J. Endoer. 8, 347 (1952). L. B. KWAKYE, A. RHIMtgR and J. VAJDA, Acts morph, neerl.scand. 9, 303 (1971/1972).
Effect of removing one ovary and a half on ovulation number and various organ weights Group
Control (N = s) One ovary and a half removed (N = ~0/
Body weight (g) :[: S.E.
Eggs ovulated per rat =[: S.E.
Organ weights (rag/100 g body wt. ~c S.E. Ovary ~
Uterus
Adrenalb
216.7 =L 2.8
9.5 • 0.6
16.1 -t- 0.8
117.5 -4- 2.8
26.1 4- 1.2
212.3 4-2.8
5.1 :t_ 0.6 c
18.8 :Jzl.4
114.3+ 4.7
30.2 zt= 1.0 o
a Mean ovarian weight is one-half of the sum of both ovaries for control rats and the weight of the remaining ovarian fi'agment for the experimental group, b Mean adrenal weight is both adrenals for all rats. o P < 0.025 when compared to value for control rats.
