Histochemistry 44, 95--101 (1975) 9 by Springer-Verlag 1975
Immunofluorescence Demonstration of Avidin in the Immature Chick Oviduct Epithelium after Progesterone Pentti Tuohimaa Institute of Biomedical Sciences, University of Tampere Tampere, Finland Received March 25, 1975
Summary. Immature chicks were used for the experiments 1-2 days after hatching. A group of chicks were injected with 5 mg of progesterone and sacrificed 1 or 24 hr later. An other group of chicks were injected daily for 9 days with 5 mg of diethylstilbestrol and thereafter with single injection of progesterone. Cryostat sections were incubated with rabbit anti-avidin serum and with fluoreseein labelled antirabbit globulin for fluorescence histochemistry. In the control animals no epithelial cells of the oviduct were fluorescence positive independently whether or not the animals were pretreated with diethylstilbestrol. One hour after administration of progesterone epithelial cells showed occasionally a slight synthesis of avidin. 24 hours after the injection of progesterone most, however never all, of the epithelial cells showed avidin in the apical part of the cell. The fluorescence reaction was clearly more intense if the animals were estrogen-primed. Diethylstilbestrol caused a differentiation of oviductal glands which were, however, only occasionally avidin positive after progesterone. These results suggest that primitive oviductal cells can proeude avidin without preceding differentiation whereas estrogen causes a differentiation of new line of cells which have regularly lost their capacity of avidin synthesis after progesterone administration. Introduction A v i d i n is a s e c r e t o r y p r o t e i n of t h e a v i a n oviduct. I t is i n d u c e d b y p r o g e s t e r o n e in i m m a t u r e a n d e s t r o g e n - p r i m e d chicks (Herz et al., 1943; O ' M a l l e y et al., 1969; K e l l o k u m p u - L e h t i n e n et al., 1975). T h e m a g n i t u d e of t h e a v i d i n s t i m u l a t i o n b y p r o g e s t e r o n e varies being higher in t h e l a t t e r ones (O'Malley et al., 1969). A v i d i n a n d e s t r o g e n - i n d u c e d proteins, e.g. o v a l b u m i n , are secreted b y s e p a r a t e cells of t h e o v i d u c t a l e p i t h e l i u m (Kohler et al., 1968). A d i f f e r e n t i a t i o n of t h e cells b y estrogen for several d a y s is r e q u i r e d for o v a l b u m i n secretion whereas a v i d i n is secreted b y t h e i m m a t u r e oviductM cells few hours a f t e r t h e a d m i n i s t r a t i o n of exogenous p r o g e s t e r o n e (O'MMley et al., 1969; K e l l o k u m p u - L e h t i n e n et al., 1975). Therefore a direct a c t i o n of p r o g e s t e r o n e on t h e m o r p h o l o g y of a v i d i n synthesis can be s t u d i e d in t h e i m m a t u r e chicks. There is considerable i n t e r e s t in t h e submicroscopic m o r p h o l o g y of progesterone action on t h e chick o v i d u c t (O'Malley et al., 1969; Oka a n d Schimke, 1969; Cox a n d Sauerwein, 1970; P M m i t e r a n d W r e n n , 1971; K e l l o k u m p u - L e h t i n e n et al., 1975). H o w e v e r , t h e a v i d i n secreting cells in t h e i m m a t u r e (not esterogen-primed) chick o v i d u c t a f t e r progesterone injection has n o t y e t been carefully identified. I n this r e p o r t i m m u n o h i s t o c h e m i c M d e m o n s t r a t i o n of those epithelial cells has been described.
Material and Methods White Leghorn Chicks (Strain Ti 53 and Ti 453, Turan Muna Hatchery, Turku, Finland) were injected 1-2 days after hatching with 5 mg of progesterone and killed 1 or 24 hr after the injection. Another group of chicks were injected with 0,5 mg of diethylstilbestrol daily
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for 9 days and 24 hr later 5 mg of progesterone was given. Controls received oil only. Oviducts were removed under ether anesthesia and frozen immediately. 6 ~zm cryostat sections were fixed in acetone at --15 ~ C for 2 min or in 4.3 per cent glutaraldehyde in cacodylate buffer at + 4 ~ C for 30 min. Some sections were processed unfixed. Antibody solution was prepared from serum of the rabbit injected weekly for 5 weeks with 1 mg of avidin and 0.5 ml of Freud's adjuvant (O'Malley and Korenman, 1967). A drop of antibody solution (antiserum diluted 1 : 1 with 0.2 M phosphate buffer pH 7.4) was pipetted on each section. Slides were then incubated at room remperature in a humidified chamber for 30 min. The sections were washed twice with an excess of the buffer. A drop of diluted (1:1) fluoreseein labelled antirabbit ~-globulin obtained from goats (Behringwerke AG, Marburg-Lahn, Germany) was applied on the section. The incubation time was 30 min. Slides were washed twice in the buffer and coated with glycerin. Fluorescence was studied immediately with Leitz Wetzlar Orthomat UV-microscope with filters PG12+PG38. Fluorescence was quite stable up to 1-2 hr. A constant exposure time of 5 rain for Kodak Tri-x, Pan 30 DIN fihn was used.
Results T h e o v i d u c t s of t h e control animals showed no b r i g h t a v i d i n fluerescence (Fig. 2 A a n d B). Similarly, t h e o v i d u c t s of e s t r o g e n - p r i m e d chicks u s u a l l y showed no avidin, b u t occassionally t h e r e were single cells in t h e epithelium w i t h slight a v i d i n fluorescence. P r o g e s t e r o n e t r e a t m e n t for one h o u r i n d u c e d no enhancem e n t in t h e fluorescence; occassional a v i d i n positive cells could be observed (Fig. 4). 24 hr after progesterone a d m i n i s t r a t i o n m o s t of t h e epithelial cells showed a v i d i n in t h e apical p a r t of cells in t h e n o n - p r i m e d animals (Fig. 1 A a n d B). I t should be n o t e d t h a t t h e r e were always some epithelial cells which were fluorescence negative. Also t h e i n t e n s i t y of t h e fluorescence v a r i e d from cell to cell. On t h e o t h e r h a n d , if t h e chicks were p r i m e d w i t h d i e t h y l s t i l b e s t r o l for 9 d a y s , t h e fluorescence was m o r e intense a n d it was l o c a t e d in all p a r t s of t h e cell (Fig. 3). Some of t h e g l a n d u l a r cells showed also fluorescence. I f t h e o v i d u e t a l sections were unfixed, m o s t of t h e fluorescence was lost. Also g l u t a r a l d e h y d e f i x a t i o n a b o l i s h e d all t h e fluorescence (:Fig. 5). T h e f i x a t i o n in cold a c e t o n e seemed to give o p t i m a l results.
Discussion The p r e s e n t results i n d i c a t e clearly t h a t a v i d i n is secreted b y t h e epithelial cells of t h e m a g n u m p a r t of t h e oviduct. The fluorescence of a v i d i n after t h e same dose of progesterone is m o r e intense when t h e chicks are estrogen-primed. This is in a g r e e m e n t w i t h t h e earlier biochemical analysis t h a t t h e m a g n i t u d e of a v i d i n i n d u c t i o n is d e p e n d e n t on t h e d u r a t i o n of estrogen p r e t r e a t m e n t (O'MMley et al., 1969). On t h e o t h e r h a n d , estrogen p r i m i n g is n o t necessary for t h e a v i d i n synthesis, because a v i d i n is d e t e c t a b l e after single injection of progesterone into t h e u n t r e a t e d chicks. I n turn, estrogen induces a proliferation of a new line of cells, g l a n d u l a r cells, which do n o t synthesize a v i d i n since t h e y are fluorescencen e g a t i v e a f t e r progesterone. These cells response m a i n l y to estrogens a n d synthesize several s e c r e t o r y proteins, e.g. o v a l b u m i n (O'Malley et al., 1969). The p o t e n t i a t i n g effect of estrogen on a v i d i n synthesis can be due to its s t i m u l a t o r y action on p r o g e s t e r o n e secretion (Elo et al., 1975). Therefore t h e epithelial cells are slightly p r o g e s t e r o n e - p r i m e d and, thus, m o r e responsive. On t h e o t h e r h a n d , estrogen s t i m u l a t e s r i b o s o m a l f o r m a t i o n in all of t h e epithelial cells leading to b e t t e r p r o t e i n synthesis c a p a c i t y (O'Malley et al., 1969).
Cytodifferentiation of Avidin Secreting Cells
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B
Fig. 1A and B. The avidin fluerescence (A) in the untreated chick oviduct 24 hr after progesterone administration (5 rag) and the same section post-stained with hematoxylin a n d eosin (B). Fixation in cold acetone. Magnification • 500 7
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A
B Fig. 2 A and B. The oviductal epithelium with no fluorescence (A) in the non-treated control chicks and the same section post-stained with hematoxylin and eosin (B). Fixation in cold acetone. Magzlification • 500
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Fig. 3. The avidin fluorescence in the diethylstilbestrol-primed (0.5 mg • 9 days) chicks 24 hr after progesterone administration. Arrows point glandular cells. Fixation in cold acetone. Magnification • 2000
4
Fig. 4. The avidin fluorescence in the untreated chick oviduct 1 hr after progesterone administration. Fixation in cold acetone. Magnification • 500
7*
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Fig. 5. The oviduetal epithdium of the untreated chick oviduct 24 hr after progesterone administration Fixation in 4.3% glutaraldehyde, magnification • 500
Several factors may hamper the results obtained in the avidin immunefluorescence. Avidin is a water-soluble protein. Therefore some of its activity was lost when the sections were incubated unfixed. On the other hand, aldehydefixation seems to destroy antigenic properties of avidin. The fluorescense was not observed when the sections were fixed in glutaraldehyde. Thus, the study of avidin immunohistoehemistry at the submicroscopic level requires dry cryo-ultramicrotome technique. The method of choice for fixation proved to be cold acetone. I t also precipitated avidin eomplitely in the test tube. Therefore, it is not probable that any avidin is lost after acetone fixation. In the control chicks there was no avidin fluorescence in the epithelial cells of the oviduct. Also the biochemical analysis shows no avidin in the non-treated animals (Kellokumpu-Lehtinen etaI., 1975). The histoehemieal observations indicate that this is due to complito absence of avidin in the epithelial cells. This assumption seems to be valid because of the several times higher sensitivity of immunofluoreseence method as an assay of avidin. Avidin can be detected in the immature chick oviducts after progesterone only if several oviducts are pooled for biochemical assay of avidin (sensitivity appr. 0.02 ~g/g of tissue) (KellokumpuLehtinen et al., 1975). However, histochemically an intense fluorescence of avidin can be detected even in a single cell of these animals. The present results confirm our previous finding that the non-treated chiek oviducts are in the secretory phase 24 hr after progesterone administration because the fluerescence is located in the apical cytoplasm and the oviduetal lumen. On the other hand, the apical location
Cytodiffercntiation of Avidin Secreting Cells
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of a v i d i n m a y i n d i c a t e t h e location of a v i d i n synthesis. Thus, t h e location of a v i d i n synthesis m a y v a r y in t h e e s t r o g e n - p r i m e d a n d estrogen u n t r e a t e d o v i d u e t a l cells. I n t h e e s t r o g e n - p r i m e d chicks a v i d i n is s y n t h e s i z e d b o t h in t h e apical a n d basal p a r t of t h e epithelial cell. E l e c t r o n microscopic observations of t h e chick o v i d u c t r e v e a l t h a t polysomes are l o c a t e d m a i n l y in t h e apical p a r t of the epit h e l i a l cell after p r o g e s t e r o n e injection only ( K e l l o k u m p u - L e h t i n e n et al., 1975), whereas t h e y are u n i v e s e l y d i s t r i b u t e d in t h e cells after estrogen priming (O'Malley et al., 1969). Some of t h e epithelial cells of u n t r e a t e d animals are a v i d i n n e g a t i v e even 24 hr a f t e r p r o g e s t e r o n e a d m i n i s t r a t i o n . This m a y be due to t h e v a r y i n g degree of p r o g e s t e r o n e s t i m u l a t i o n in different cells. On t h e o t h e r hand, these cells m a y also be t h e p r o g e n i t o r cells of t h e estrogen-induced a v i d i n - n e g a t i v e g l a n d u l a r cells.
Aclcnowledgements. This work was supported by a grant (No M 73.80) from the Population Council, New York, U.S.A.
References Cox, R. F., Sauerwein, H. : Studies on the mode of action of progesterone on chicken oviduct epithelium I. Morphological changes assiciated with early differentiation of the tissue. Exp. Cell Res. 61, 7940 (1970) Elo, H., Tuohimaa, P., Jgnne, O. : Cumulative superinduction of avidin in the chick oviduct by tissue damage and actinomycin D injections. ~olec. Cell. Endocr. 2, 203-211 (1975) Hertz, R., Fraps, R. lVI., Sebrell, W. H. : Induction of avidin formation in the avian oviduct by stilbestrol plus progesterone. Proc. Soc. exp. Biol. (N.Y.) 52, 142-144 (1943) Kellokumpu-Lehtinen, P., Jokelainen, P. T., Tuohimaa, P. : Early eytodifferentiation of the chick oviduct epithelium by progesterone. J. Ultrastruct. 1Res. (1975) Kohler, P. 0., Grimley, P. M., O'Malley, B. W. : Estrogen-induced cytodifferentiation of the ovalbumin-secreting glands of the chick oviduct. J. Cell Biol. 40, 8-27 (1969) Oka, T., Schimke, R. T. : Progesterone antagonism of estrogen-induced cytodiffcrentiation in chick oviduct. Science 163, 83-85 (1969) O'Malley, B. W., Korenman, S. G. : Studies on the mechanism of hormone induction of a specific protein. Immunological identity and kinetic studies of avidin synthesized in vitro by the chick oviduct. Life Sci. 6, 1953-1969 (1967) O'Malley, B. W., McGuire, W. L., Kohler, P. 0., Korenman, S. G.: Studies on the mechanism of steroid hormone regulation of synthesis of specific proteins. Recent Progr. Hormone Res. 2~, 105-160 (1969) Palmiter, R. D., Wrenn, J . T . : Interaction of estrogen and progesterone in chick oviduct development 3: Tubular gland cell cytodifferentiation. J. Cell Biol. 50, 598-615 (1971) Prof. Dr. P. Tuohimaa Univ. of Tampere Institute of Biomedical Sciences SF-33520 Tampere 52/Finland
Immunofluorescence Demonstration of Avidin in the Immature Chick Oviduct Epithelium after Progesterone Pentti Tuohimaa Institute of Biomedical Sciences, University of Tampere Tampere, Finland Received March 25, 1975
Summary. Immature chicks were used for the experiments 1-2 days after hatching. A group of chicks were injected with 5 mg of progesterone and sacrificed 1 or 24 hr later. An other group of chicks were injected daily for 9 days with 5 mg of diethylstilbestrol and thereafter with single injection of progesterone. Cryostat sections were incubated with rabbit anti-avidin serum and with fluoreseein labelled antirabbit globulin for fluorescence histochemistry. In the control animals no epithelial cells of the oviduct were fluorescence positive independently whether or not the animals were pretreated with diethylstilbestrol. One hour after administration of progesterone epithelial cells showed occasionally a slight synthesis of avidin. 24 hours after the injection of progesterone most, however never all, of the epithelial cells showed avidin in the apical part of the cell. The fluorescence reaction was clearly more intense if the animals were estrogen-primed. Diethylstilbestrol caused a differentiation of oviductal glands which were, however, only occasionally avidin positive after progesterone. These results suggest that primitive oviductal cells can proeude avidin without preceding differentiation whereas estrogen causes a differentiation of new line of cells which have regularly lost their capacity of avidin synthesis after progesterone administration. Introduction A v i d i n is a s e c r e t o r y p r o t e i n of t h e a v i a n oviduct. I t is i n d u c e d b y p r o g e s t e r o n e in i m m a t u r e a n d e s t r o g e n - p r i m e d chicks (Herz et al., 1943; O ' M a l l e y et al., 1969; K e l l o k u m p u - L e h t i n e n et al., 1975). T h e m a g n i t u d e of t h e a v i d i n s t i m u l a t i o n b y p r o g e s t e r o n e varies being higher in t h e l a t t e r ones (O'Malley et al., 1969). A v i d i n a n d e s t r o g e n - i n d u c e d proteins, e.g. o v a l b u m i n , are secreted b y s e p a r a t e cells of t h e o v i d u c t a l e p i t h e l i u m (Kohler et al., 1968). A d i f f e r e n t i a t i o n of t h e cells b y estrogen for several d a y s is r e q u i r e d for o v a l b u m i n secretion whereas a v i d i n is secreted b y t h e i m m a t u r e oviductM cells few hours a f t e r t h e a d m i n i s t r a t i o n of exogenous p r o g e s t e r o n e (O'MMley et al., 1969; K e l l o k u m p u - L e h t i n e n et al., 1975). Therefore a direct a c t i o n of p r o g e s t e r o n e on t h e m o r p h o l o g y of a v i d i n synthesis can be s t u d i e d in t h e i m m a t u r e chicks. There is considerable i n t e r e s t in t h e submicroscopic m o r p h o l o g y of progesterone action on t h e chick o v i d u c t (O'Malley et al., 1969; Oka a n d Schimke, 1969; Cox a n d Sauerwein, 1970; P M m i t e r a n d W r e n n , 1971; K e l l o k u m p u - L e h t i n e n et al., 1975). H o w e v e r , t h e a v i d i n secreting cells in t h e i m m a t u r e (not esterogen-primed) chick o v i d u c t a f t e r progesterone injection has n o t y e t been carefully identified. I n this r e p o r t i m m u n o h i s t o c h e m i c M d e m o n s t r a t i o n of those epithelial cells has been described.
Material and Methods White Leghorn Chicks (Strain Ti 53 and Ti 453, Turan Muna Hatchery, Turku, Finland) were injected 1-2 days after hatching with 5 mg of progesterone and killed 1 or 24 hr after the injection. Another group of chicks were injected with 0,5 mg of diethylstilbestrol daily
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for 9 days and 24 hr later 5 mg of progesterone was given. Controls received oil only. Oviducts were removed under ether anesthesia and frozen immediately. 6 ~zm cryostat sections were fixed in acetone at --15 ~ C for 2 min or in 4.3 per cent glutaraldehyde in cacodylate buffer at + 4 ~ C for 30 min. Some sections were processed unfixed. Antibody solution was prepared from serum of the rabbit injected weekly for 5 weeks with 1 mg of avidin and 0.5 ml of Freud's adjuvant (O'Malley and Korenman, 1967). A drop of antibody solution (antiserum diluted 1 : 1 with 0.2 M phosphate buffer pH 7.4) was pipetted on each section. Slides were then incubated at room remperature in a humidified chamber for 30 min. The sections were washed twice with an excess of the buffer. A drop of diluted (1:1) fluoreseein labelled antirabbit ~-globulin obtained from goats (Behringwerke AG, Marburg-Lahn, Germany) was applied on the section. The incubation time was 30 min. Slides were washed twice in the buffer and coated with glycerin. Fluorescence was studied immediately with Leitz Wetzlar Orthomat UV-microscope with filters PG12+PG38. Fluorescence was quite stable up to 1-2 hr. A constant exposure time of 5 rain for Kodak Tri-x, Pan 30 DIN fihn was used.
Results T h e o v i d u c t s of t h e control animals showed no b r i g h t a v i d i n fluerescence (Fig. 2 A a n d B). Similarly, t h e o v i d u c t s of e s t r o g e n - p r i m e d chicks u s u a l l y showed no avidin, b u t occassionally t h e r e were single cells in t h e epithelium w i t h slight a v i d i n fluorescence. P r o g e s t e r o n e t r e a t m e n t for one h o u r i n d u c e d no enhancem e n t in t h e fluorescence; occassional a v i d i n positive cells could be observed (Fig. 4). 24 hr after progesterone a d m i n i s t r a t i o n m o s t of t h e epithelial cells showed a v i d i n in t h e apical p a r t of cells in t h e n o n - p r i m e d animals (Fig. 1 A a n d B). I t should be n o t e d t h a t t h e r e were always some epithelial cells which were fluorescence negative. Also t h e i n t e n s i t y of t h e fluorescence v a r i e d from cell to cell. On t h e o t h e r h a n d , if t h e chicks were p r i m e d w i t h d i e t h y l s t i l b e s t r o l for 9 d a y s , t h e fluorescence was m o r e intense a n d it was l o c a t e d in all p a r t s of t h e cell (Fig. 3). Some of t h e g l a n d u l a r cells showed also fluorescence. I f t h e o v i d u e t a l sections were unfixed, m o s t of t h e fluorescence was lost. Also g l u t a r a l d e h y d e f i x a t i o n a b o l i s h e d all t h e fluorescence (:Fig. 5). T h e f i x a t i o n in cold a c e t o n e seemed to give o p t i m a l results.
Discussion The p r e s e n t results i n d i c a t e clearly t h a t a v i d i n is secreted b y t h e epithelial cells of t h e m a g n u m p a r t of t h e oviduct. The fluorescence of a v i d i n after t h e same dose of progesterone is m o r e intense when t h e chicks are estrogen-primed. This is in a g r e e m e n t w i t h t h e earlier biochemical analysis t h a t t h e m a g n i t u d e of a v i d i n i n d u c t i o n is d e p e n d e n t on t h e d u r a t i o n of estrogen p r e t r e a t m e n t (O'MMley et al., 1969). On t h e o t h e r h a n d , estrogen p r i m i n g is n o t necessary for t h e a v i d i n synthesis, because a v i d i n is d e t e c t a b l e after single injection of progesterone into t h e u n t r e a t e d chicks. I n turn, estrogen induces a proliferation of a new line of cells, g l a n d u l a r cells, which do n o t synthesize a v i d i n since t h e y are fluorescencen e g a t i v e a f t e r progesterone. These cells response m a i n l y to estrogens a n d synthesize several s e c r e t o r y proteins, e.g. o v a l b u m i n (O'Malley et al., 1969). The p o t e n t i a t i n g effect of estrogen on a v i d i n synthesis can be due to its s t i m u l a t o r y action on p r o g e s t e r o n e secretion (Elo et al., 1975). Therefore t h e epithelial cells are slightly p r o g e s t e r o n e - p r i m e d and, thus, m o r e responsive. On t h e o t h e r h a n d , estrogen s t i m u l a t e s r i b o s o m a l f o r m a t i o n in all of t h e epithelial cells leading to b e t t e r p r o t e i n synthesis c a p a c i t y (O'Malley et al., 1969).
Cytodifferentiation of Avidin Secreting Cells
97
B
Fig. 1A and B. The avidin fluerescence (A) in the untreated chick oviduct 24 hr after progesterone administration (5 rag) and the same section post-stained with hematoxylin a n d eosin (B). Fixation in cold acetone. Magnification • 500 7
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A
B Fig. 2 A and B. The oviductal epithelium with no fluorescence (A) in the non-treated control chicks and the same section post-stained with hematoxylin and eosin (B). Fixation in cold acetone. Magzlification • 500
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Fig. 3. The avidin fluorescence in the diethylstilbestrol-primed (0.5 mg • 9 days) chicks 24 hr after progesterone administration. Arrows point glandular cells. Fixation in cold acetone. Magnification • 2000
4
Fig. 4. The avidin fluorescence in the untreated chick oviduct 1 hr after progesterone administration. Fixation in cold acetone. Magnification • 500
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Fig. 5. The oviduetal epithdium of the untreated chick oviduct 24 hr after progesterone administration Fixation in 4.3% glutaraldehyde, magnification • 500
Several factors may hamper the results obtained in the avidin immunefluorescence. Avidin is a water-soluble protein. Therefore some of its activity was lost when the sections were incubated unfixed. On the other hand, aldehydefixation seems to destroy antigenic properties of avidin. The fluorescense was not observed when the sections were fixed in glutaraldehyde. Thus, the study of avidin immunohistoehemistry at the submicroscopic level requires dry cryo-ultramicrotome technique. The method of choice for fixation proved to be cold acetone. I t also precipitated avidin eomplitely in the test tube. Therefore, it is not probable that any avidin is lost after acetone fixation. In the control chicks there was no avidin fluorescence in the epithelial cells of the oviduct. Also the biochemical analysis shows no avidin in the non-treated animals (Kellokumpu-Lehtinen etaI., 1975). The histoehemieal observations indicate that this is due to complito absence of avidin in the epithelial cells. This assumption seems to be valid because of the several times higher sensitivity of immunofluoreseence method as an assay of avidin. Avidin can be detected in the immature chick oviducts after progesterone only if several oviducts are pooled for biochemical assay of avidin (sensitivity appr. 0.02 ~g/g of tissue) (KellokumpuLehtinen et al., 1975). However, histochemically an intense fluorescence of avidin can be detected even in a single cell of these animals. The present results confirm our previous finding that the non-treated chiek oviducts are in the secretory phase 24 hr after progesterone administration because the fluerescence is located in the apical cytoplasm and the oviduetal lumen. On the other hand, the apical location
Cytodiffercntiation of Avidin Secreting Cells
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of a v i d i n m a y i n d i c a t e t h e location of a v i d i n synthesis. Thus, t h e location of a v i d i n synthesis m a y v a r y in t h e e s t r o g e n - p r i m e d a n d estrogen u n t r e a t e d o v i d u e t a l cells. I n t h e e s t r o g e n - p r i m e d chicks a v i d i n is s y n t h e s i z e d b o t h in t h e apical a n d basal p a r t of t h e epithelial cell. E l e c t r o n microscopic observations of t h e chick o v i d u c t r e v e a l t h a t polysomes are l o c a t e d m a i n l y in t h e apical p a r t of the epit h e l i a l cell after p r o g e s t e r o n e injection only ( K e l l o k u m p u - L e h t i n e n et al., 1975), whereas t h e y are u n i v e s e l y d i s t r i b u t e d in t h e cells after estrogen priming (O'Malley et al., 1969). Some of t h e epithelial cells of u n t r e a t e d animals are a v i d i n n e g a t i v e even 24 hr a f t e r p r o g e s t e r o n e a d m i n i s t r a t i o n . This m a y be due to t h e v a r y i n g degree of p r o g e s t e r o n e s t i m u l a t i o n in different cells. On t h e o t h e r hand, these cells m a y also be t h e p r o g e n i t o r cells of t h e estrogen-induced a v i d i n - n e g a t i v e g l a n d u l a r cells.
Aclcnowledgements. This work was supported by a grant (No M 73.80) from the Population Council, New York, U.S.A.
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