ZIPGRAM
RAPID COMMUNICATION
LOCALIZATION OF ANTISERA TO LHB AND FSHB IN THE RAT PITUITARY GLAND (1) DAMON C. HERBERT Department of Anatomy, The U n i v e r s i t y of Texas Health S c i e n c e Center a t San Antonio, San Antonio, Texas 78284
ABSTRACT P i t u i t a r y glands from a d u l t male and female r a t s were s t a i n e d by the immunocytochemical b r i d g e t e c h n i q u e w i t h a n t i s e r a t o o v i n e L H B and human FSHB. Peroxidase r e a c t i o n product was l o c a l i z e d i n t h e same PAS-positive c e l l s w i t h both a n t i s e r a . The c e l l s v a r i e d i n s i z e and topographical d i s t r i b u t i o n w i t h i n the p a r s d i s t a l i s . These o b s e r v a t i o n s s u p p o r t the concept t h a t a s i n g l e c e l l type i s r e s p o n s i b l e f o r the s y n t h e s i s o f both L H and FSH i n t h e r a t . For a number of y e a r s i t was b e l i e v e d t h a t the gonadotrophins, LH and FSH, were produced i n s e p a r a t e and d i s t i n c t p i t u i t a r y c e l l t y p e s .
Using
d i f f e r e n t i a l l i g h t microscopic s t a i n i n g t e c h n i q u e s , Pur-ves and Griesbach ( ' 5 4 ) and Rennels ( ' 5 7 ) were s u c c e s s f u l i n d i f f e r e n t i a t i n g two t y p e s of
gonadotrophs.
These c e l l s were p r e s e n t i n s e p a r a t e r e g i o n s of t h e r a t
hypophysis and were considered as being f u n c t i o n a l l y d i f f e r e n t based on
studies i n the c a s t r a t e d animal (Purves and Griesbach, ' 5 5 ; Hildebrand e t a l . , '57).
Using the immunofluorescent antibody t e c h n i q u e , one of t h e gona-
dotrophs was shown t o c o n t a i n LH and the o t h e r FSH i n o v i n e ( R e n n e l s , ' 6 3 ) and r a t ( S h i i n o and Rennels, ' 6 8 ) p i t u i t a r y g l a n d s .
Two gonadotrophic c e l l -
t y p e s have been d i s t i n g u i s h e d by e l e c t r o n microscopy, based on their u l t r a s t r u c t u r a l appearance (Kurosumi , ' 6 8 ) , which do not undergo the same morphol o g i c a l changes following exogenous a d m i n i s t r a t i o n o f gonadotrophin-releasi n g hormone ( S h i i n o e t a l . , ' 7 2 ) . 379
The one cell-one hormone theory w i t h respect t o t h e gonadotrophs and LH and FSH has not been universally accepted.
Following examination
of pi t u i t a r i es extracted with tri chloroaceti c acid , Barrnett e t a1 . ( I 56) proposed the idea t h a t these two hormones may be secreted by a s i n g l e c e l l type.
Marked and q u i t e s i m i l a r u l t r a s t r u c t u r a l changes were described
i n a l l gonadotrophs following c a s t r a t i o n in the rat (Yoshimura and Harumiya, ' 6 5 ) .
Using immunocytochemical techniques, LH and FSH have been
shown t o be present i n the same c e l l in t h e p i t u i t a r y of t h e r a t (Nakane, '70) and the human (Phifer et a l . , ' 7 3 ) . I t i s generally accepted t h a t t h e s p e c i f i c immunologic a c t i v i t y o f the gonadotrophins resides i n t h e beta s u b u n i t of each hormone.
In t h e
l i g h t of t h i s , however, no one has reported using immunocytochemistry with a n t i s e r a t o both LHB and FSHB i n an e f f o r t t o determine whether or not t h e gonadotrophins a r e present in a s i n g l e population of p i t u i t a r y cells.
Since we have recently prepared such a n t i s e r a i n our laboratory,
t h e present investigation was c a r r i e d out i n an attempt t o resolve t h i s probl em. MATERIALS AND METHODS P i t u i t a r y glands were removed from f i v e a d u l t male and from f i v e a d u l t female Sprague-Dawley r a t s following decapitat i o n , and were fixed f o r six hours i n formol-sublimate o r Bouin's solution and embedded i n paraffin.
No fewer than 25 t i s s u e sections from each of
t h e animals were stained by the immunocytochemical bridge technique or by the trichrome procedure of El Etreby e t a l . ( ' 7 2 ) .
The hormone-specific
a n t i s e r a were prepared i n a d u l t male r a t s against ovine LHB (HSN-6-82B2) o r human FSHB (LER-1817-2B).
During each immunization, The animals re-
ceived several subcutaneous i n j e c t i o n s o f the hormone suspended in complete Freund's adjuvant along w i t h 0.5cc o f Bordetella p e r t u s s i s vaccine.
380
All
a n t i s e r a were absorbed prior t o t h e i r use with normal serum p r o t e i n s , the homologous alpha subunit, ACTH, GH and TSH.
Control experiments s i m i l a r
t o those described by Petrusz ( ' 7 4 ) were c a r r i e d out t o assure s p e c i f i c i t y of s t a i n i n g . RESULTS The identical c e l l was stained with t h e a n t i s e r a t o LHB and FSHB ( f i g s . l a , b ) i n both t h e female and male r a t p i t u i t a r y glands.
This
observation was consistently found i n t i s s u e sections taken from repres e n t a t i v e areas of the e n t i r e pars d i s t a l i s .
The immunocytochemical s t a i n -
ing was present i n two d i f f e r e n t s i z e s of c e l l s which were generally ovoid in shape, although t h i s was somewhat variable, e s p e c i a l l y i n females.
The
smaller sized gonadotrophs were observed mainly towards the periphery of the a n t e r i o r lobe and i n the central and ventral regions, while the l a r g e r gonadotrophs were primarily located in the "sex zone," adjacent t o the p a r s intermedia and i n t h e central and l a t e r a l regions of the pars d i s t a l i s . The l a r g e r gonadotrophs appeared t o be polarized and frequently displayed a negative image of the Golgi apparatus.
They a l s o seemed t o outnumber
the smaller gonadotrophs in p i t u i t a r i e s from male as well as female r a t s . Neither the small nor the l a r g e gonadotrophic c e l l s were immunocytochemi c a l l y stained with antiserum t o human TSHB.
Instead, the antiserum re-
acted with a small, angular c e l l t h a t was located principally within the more central regions of t h e pars d i s t a l i s .
When the p i t u i t a r y glands were
stained with a l c i a n blue, PAS and orange G , the gonadotrophs stained posi t i v e w t h PAS and t h e thyrotrophs w i t h a l c i a n blue. A1
imnunocytochemical s t a i n i n g was prevented following absorption of
each antiserum with i t s homologous antigen.
Preincubation of 1 ml of the
anti-LHB serum w i t h 1 mg of ovine FSH o r 1 ml of the anti-FSHB serum w i t h 1 mg of HCG did not a f f e c t t h e s t a i n i n g .
381
Likewise, absorption of 1 ml of
the anti-TSHB serum with 1 mg of HCG o r human FSHB f a i l e d t o remove the i mmu no 1 og i cal s t ai n i ng
.
DISCUSSION These findings demonstrate the presence of a single population of PAS-positive c e l l s , present within the pars d i s t a l i s o f the r a t , t h a t contain both LH and FSH.
These c e l l s d i f f e r from the thyrotrophs
in t h e i r lack of s t a i n a b i l i t y with alcian blue, t h e i r topographical dist r i b u t i o n within the pars d i s t a l i s and the f a c t t h a t they do not s t a i n immunochemically with a n t i s e r a t o human TSHB.
They a r e distributed in re-
gions of the pars d i s t a l i s t h a t have been described as being populated by the gonadotrophs (Purves and Griesbach, ' 5 4 ; Rennels , ' 5 7 ) and appear t c be the same c e l l s t h a t Baker e t a l . ( ' 7 2 ) stained with anti-LHB serum. The findings i n the present report a r e supported by the recent observations of Moriarty ( ' 75) who observed immunochemical s t a i n i n g a t the ul t r a s t r u c tural level in b o t h the LH and t h e FSH c e l l s in the female r a t using a n t i sera t o LH.
Additionally, analysis of selected regions of t h e pars d i s t a l i s
of the rat by radioimmunoassay demonstrated no striking differences in the r a t i o of LH t o FSH in any portion of the gland (Schafer and McShan, ' 7 4 ) . Although the observations made in t h i s report c l e a r l y demonstrate t h a t LH and FSH a r e present i n the same population of c e l l s , an additional in-
t e r p r e t a t i o n of t h e findings might be made.
I t i s possible t h a t LHB a n d
FSHB share enough common antigenic determinants so t h a t a n t i s e r a generated
FIGURE LEGEND 1A-D Adjacent 3 urn-thick s e r i a l sections from an a d u l t female ( A , B ) and a n adult male (C,D) r a t p i t u i t a r y gland following immunocytochemical s t a i n i n g with anti-LHB serum ( A , C ) and anti-FSHB serum ( B , D ) . The identical c e l l s a r e s t a i n i n g with b o t h a n t i s e r a . A few c e l l s in each p a i r o f micrographs a r e labelled f o r comparison. X775 ( A , B ) ; X l O O O ( C , D ) .
382
against these subunits a r e incapable of d i f f e r e n t i a t i n g t h e LH and FSH c e l l by immunocytochemistry.
Thus, the two gonadotrophins may indeed be
produced by separate p i t u i t a r y c e l l types; however, techniques other than immunocytochemistry may have t o be employed i n order t o verffy t h i s . ACKNOWLEDGMENTS The author wishes t o thank Drs. L . Reichert and D.
Ward f o r providing us with t h e hormone preparations, Drs. E. G . Rennels,
M. Shiino and P . Bowie f o r t h e i r advice during t h e course of t h i s invest i g a t i o n , and Miss P. Cisneros and Mrs. M. C a s t i l l e j a f o r t h e i r technical assistance. LITERATURE CITED Baker, B. L . , J . G. Pierceand J . S. Cornell 1972 The u t i l i t y of antiserums t o subunits of TSH and LH f o r immunochemical staining of the r a t hypophysis. h. J . Anat., 135: 251-268. Barrnett, R. J . , E. J. Ladman, N . J . McAllaster and E . R. Siperstein 1956 The localization of glycoprotein hormones i n the a n t e r i o r p i t u i t a r y glands of r a t s investigated by d i f f e r e n t i a l protein s o l u b i l i t i e s , histological s t a i n s and bio-assays. Endocrinology, 59: 398-418. Etreby, M. F., K.-D. Richter and P. Gunzel 1972 Histological and histochemical d i f f e r e n t i a t i o n of the glandular c e l l s of the a n t e r i o r p i t u i t a r y i n various experimental animals. In: Toxicology: Review and Prospect. I n t . Congr. Series No. 219. W . A . M. Duncan, ed. Excerpta Medica, Amsterdam, pp. 270-281. ldebrand, J . E., E. G. Rennels and J. C. Finerty 1957 Gonadotrophic c e l l s of t h e r a t hypophysis and t h e i r r e l a t i o n t o hormone production. Z. Zellforsch. Mikrosk. Anat., 46: 400-411. Kurosumi, K. 1968 Functional c l a s s i f i c a t i o n of c e l l types of t h e a n t e r i o r p i t u i t a r y gland accomplished by electron microscopy. Arch. h i s t o l . j a p . , 29: 329-362. Moriarty, G. C. 1975 Subcellular l o c a l i z a t i o n of l u t e i n i z i n g hormone i n t h e r a t a n t e r i o r p i t u i t a r y : a sex difference in the morphology a n d cytochemistry of LH gonadotrophs. Anat. Rec., 181: 430 (Abstract). Nakane, P. K. 1970 Classifications of a n t e r i o r p i t u i t a r y cell types w i t h imunoenzyme histochemistry. J . Histochem. Cytochem., 18: 9-20. Petrusz, P. 1974 Demonstration of gonadotropin binding s i t e s in the r a t ovary by an immunoglobulin-enzyme bridge method. Europ. J . Obstet. Gynecol. Reprod. Biol. ( S u p p l . ) , 4: S3-S9. Phifer, R. F., A . R. Midgley and S. S. Spicer 1973 Immunohistologic and h i s t o l o g i c evidence t h a t f o l l i c l e - s t i m u l a t i n g hormone and l u t e i n i z i n g hormone a r e present in the same c e l l type i n the human pars d i s t a l i s . J . Clin. Endocrinol. Metab., 36: 125-141. 384
Purves, H . D . , and W. H . Griesbach 1954 The s i t e of f o l l i c l e s t i m u l a t i n g and l u t e i n i z i n g hormone production i n the r a t p i t u i t a r y . Endocrinology, 55: 785-793. Purves, H . D . , and W. H. Griesbach 1955 Changes i n the gonadotrophs of the r a t p i t u i t a r y a f t e r gonadectomy. Endocrinology, 56: 374-386. Rennels, E. G. 1957 Two t i n c t o r i a l types of gonadotrophic c e l l s i n t h e r a t hypophysis. Z. Zellforsch. Mikrosk. Anat., 45: 464-471. Rennels, E. G. 1963 Gonadotrophic c e l l s o f t h e r a t hypophysis. In: Cytologie de 1 'Adenohyophyse. J . Benoit and C. DeLage, eds. C. N . R. S. , P a r i s , pp. 201-213. Schafer, S. J . , and W. H. McShan 1974 Radioimnunoassay d e t e r minations o f gonadotrophic hormone content i n d i f f e r e n t regions o f male and female r a t adenohypophyses. Proc. SOC. Exp. Biol. Med., 146: 546-548. Shiino, M., A. Arimura, A. V. Schally and E . G. Rennels 1972 Ultras t r u c t u r a l observations of granule extrusion from r a t a n t e r i o r p i t u i t a r y c e l l s a f t e r i n j e c t i o n of LH-releasing hormone. Z. Zellforsch. Mikrosk. Anat. , 128: 152-161. S h i i n o , M. , and E . G. Rennels 1968 Immunohistological observations of gonadotrophs i n the a n t e r i o r p i t u i t a r y gland of t h e r a t . Jap. J . Zootech. S c i . , 12: 505-510. Yoshimura, F., and K. Harumiya 1965 Electron microscopy of the a n t e r i o r lobe of the p i t u i t a r y i n normal and c a s t r a t e d rats. Endocrinol. Japan. , 12: 119-151. RE FE REN CE 1 Supported by NIH General Research Support Grant RR05654.
385
RAPID COMMUNICATION
LOCALIZATION OF ANTISERA TO LHB AND FSHB IN THE RAT PITUITARY GLAND (1) DAMON C. HERBERT Department of Anatomy, The U n i v e r s i t y of Texas Health S c i e n c e Center a t San Antonio, San Antonio, Texas 78284
ABSTRACT P i t u i t a r y glands from a d u l t male and female r a t s were s t a i n e d by the immunocytochemical b r i d g e t e c h n i q u e w i t h a n t i s e r a t o o v i n e L H B and human FSHB. Peroxidase r e a c t i o n product was l o c a l i z e d i n t h e same PAS-positive c e l l s w i t h both a n t i s e r a . The c e l l s v a r i e d i n s i z e and topographical d i s t r i b u t i o n w i t h i n the p a r s d i s t a l i s . These o b s e r v a t i o n s s u p p o r t the concept t h a t a s i n g l e c e l l type i s r e s p o n s i b l e f o r the s y n t h e s i s o f both L H and FSH i n t h e r a t . For a number of y e a r s i t was b e l i e v e d t h a t the gonadotrophins, LH and FSH, were produced i n s e p a r a t e and d i s t i n c t p i t u i t a r y c e l l t y p e s .
Using
d i f f e r e n t i a l l i g h t microscopic s t a i n i n g t e c h n i q u e s , Pur-ves and Griesbach ( ' 5 4 ) and Rennels ( ' 5 7 ) were s u c c e s s f u l i n d i f f e r e n t i a t i n g two t y p e s of
gonadotrophs.
These c e l l s were p r e s e n t i n s e p a r a t e r e g i o n s of t h e r a t
hypophysis and were considered as being f u n c t i o n a l l y d i f f e r e n t based on
studies i n the c a s t r a t e d animal (Purves and Griesbach, ' 5 5 ; Hildebrand e t a l . , '57).
Using the immunofluorescent antibody t e c h n i q u e , one of t h e gona-
dotrophs was shown t o c o n t a i n LH and the o t h e r FSH i n o v i n e ( R e n n e l s , ' 6 3 ) and r a t ( S h i i n o and Rennels, ' 6 8 ) p i t u i t a r y g l a n d s .
Two gonadotrophic c e l l -
t y p e s have been d i s t i n g u i s h e d by e l e c t r o n microscopy, based on their u l t r a s t r u c t u r a l appearance (Kurosumi , ' 6 8 ) , which do not undergo the same morphol o g i c a l changes following exogenous a d m i n i s t r a t i o n o f gonadotrophin-releasi n g hormone ( S h i i n o e t a l . , ' 7 2 ) . 379
The one cell-one hormone theory w i t h respect t o t h e gonadotrophs and LH and FSH has not been universally accepted.
Following examination
of pi t u i t a r i es extracted with tri chloroaceti c acid , Barrnett e t a1 . ( I 56) proposed the idea t h a t these two hormones may be secreted by a s i n g l e c e l l type.
Marked and q u i t e s i m i l a r u l t r a s t r u c t u r a l changes were described
i n a l l gonadotrophs following c a s t r a t i o n in the rat (Yoshimura and Harumiya, ' 6 5 ) .
Using immunocytochemical techniques, LH and FSH have been
shown t o be present i n the same c e l l in t h e p i t u i t a r y of t h e r a t (Nakane, '70) and the human (Phifer et a l . , ' 7 3 ) . I t i s generally accepted t h a t t h e s p e c i f i c immunologic a c t i v i t y o f the gonadotrophins resides i n t h e beta s u b u n i t of each hormone.
In t h e
l i g h t of t h i s , however, no one has reported using immunocytochemistry with a n t i s e r a t o both LHB and FSHB i n an e f f o r t t o determine whether or not t h e gonadotrophins a r e present in a s i n g l e population of p i t u i t a r y cells.
Since we have recently prepared such a n t i s e r a i n our laboratory,
t h e present investigation was c a r r i e d out i n an attempt t o resolve t h i s probl em. MATERIALS AND METHODS P i t u i t a r y glands were removed from f i v e a d u l t male and from f i v e a d u l t female Sprague-Dawley r a t s following decapitat i o n , and were fixed f o r six hours i n formol-sublimate o r Bouin's solution and embedded i n paraffin.
No fewer than 25 t i s s u e sections from each of
t h e animals were stained by the immunocytochemical bridge technique or by the trichrome procedure of El Etreby e t a l . ( ' 7 2 ) .
The hormone-specific
a n t i s e r a were prepared i n a d u l t male r a t s against ovine LHB (HSN-6-82B2) o r human FSHB (LER-1817-2B).
During each immunization, The animals re-
ceived several subcutaneous i n j e c t i o n s o f the hormone suspended in complete Freund's adjuvant along w i t h 0.5cc o f Bordetella p e r t u s s i s vaccine.
380
All
a n t i s e r a were absorbed prior t o t h e i r use with normal serum p r o t e i n s , the homologous alpha subunit, ACTH, GH and TSH.
Control experiments s i m i l a r
t o those described by Petrusz ( ' 7 4 ) were c a r r i e d out t o assure s p e c i f i c i t y of s t a i n i n g . RESULTS The identical c e l l was stained with t h e a n t i s e r a t o LHB and FSHB ( f i g s . l a , b ) i n both t h e female and male r a t p i t u i t a r y glands.
This
observation was consistently found i n t i s s u e sections taken from repres e n t a t i v e areas of the e n t i r e pars d i s t a l i s .
The immunocytochemical s t a i n -
ing was present i n two d i f f e r e n t s i z e s of c e l l s which were generally ovoid in shape, although t h i s was somewhat variable, e s p e c i a l l y i n females.
The
smaller sized gonadotrophs were observed mainly towards the periphery of the a n t e r i o r lobe and i n the central and ventral regions, while the l a r g e r gonadotrophs were primarily located in the "sex zone," adjacent t o the p a r s intermedia and i n t h e central and l a t e r a l regions of the pars d i s t a l i s . The l a r g e r gonadotrophs appeared t o be polarized and frequently displayed a negative image of the Golgi apparatus.
They a l s o seemed t o outnumber
the smaller gonadotrophs in p i t u i t a r i e s from male as well as female r a t s . Neither the small nor the l a r g e gonadotrophic c e l l s were immunocytochemi c a l l y stained with antiserum t o human TSHB.
Instead, the antiserum re-
acted with a small, angular c e l l t h a t was located principally within the more central regions of t h e pars d i s t a l i s .
When the p i t u i t a r y glands were
stained with a l c i a n blue, PAS and orange G , the gonadotrophs stained posi t i v e w t h PAS and t h e thyrotrophs w i t h a l c i a n blue. A1
imnunocytochemical s t a i n i n g was prevented following absorption of
each antiserum with i t s homologous antigen.
Preincubation of 1 ml of the
anti-LHB serum w i t h 1 mg of ovine FSH o r 1 ml of the anti-FSHB serum w i t h 1 mg of HCG did not a f f e c t t h e s t a i n i n g .
381
Likewise, absorption of 1 ml of
the anti-TSHB serum with 1 mg of HCG o r human FSHB f a i l e d t o remove the i mmu no 1 og i cal s t ai n i ng
.
DISCUSSION These findings demonstrate the presence of a single population of PAS-positive c e l l s , present within the pars d i s t a l i s o f the r a t , t h a t contain both LH and FSH.
These c e l l s d i f f e r from the thyrotrophs
in t h e i r lack of s t a i n a b i l i t y with alcian blue, t h e i r topographical dist r i b u t i o n within the pars d i s t a l i s and the f a c t t h a t they do not s t a i n immunochemically with a n t i s e r a t o human TSHB.
They a r e distributed in re-
gions of the pars d i s t a l i s t h a t have been described as being populated by the gonadotrophs (Purves and Griesbach, ' 5 4 ; Rennels , ' 5 7 ) and appear t c be the same c e l l s t h a t Baker e t a l . ( ' 7 2 ) stained with anti-LHB serum. The findings i n the present report a r e supported by the recent observations of Moriarty ( ' 75) who observed immunochemical s t a i n i n g a t the ul t r a s t r u c tural level in b o t h the LH and t h e FSH c e l l s in the female r a t using a n t i sera t o LH.
Additionally, analysis of selected regions of t h e pars d i s t a l i s
of the rat by radioimmunoassay demonstrated no striking differences in the r a t i o of LH t o FSH in any portion of the gland (Schafer and McShan, ' 7 4 ) . Although the observations made in t h i s report c l e a r l y demonstrate t h a t LH and FSH a r e present i n the same population of c e l l s , an additional in-
t e r p r e t a t i o n of t h e findings might be made.
I t i s possible t h a t LHB a n d
FSHB share enough common antigenic determinants so t h a t a n t i s e r a generated
FIGURE LEGEND 1A-D Adjacent 3 urn-thick s e r i a l sections from an a d u l t female ( A , B ) and a n adult male (C,D) r a t p i t u i t a r y gland following immunocytochemical s t a i n i n g with anti-LHB serum ( A , C ) and anti-FSHB serum ( B , D ) . The identical c e l l s a r e s t a i n i n g with b o t h a n t i s e r a . A few c e l l s in each p a i r o f micrographs a r e labelled f o r comparison. X775 ( A , B ) ; X l O O O ( C , D ) .
382
against these subunits a r e incapable of d i f f e r e n t i a t i n g t h e LH and FSH c e l l by immunocytochemistry.
Thus, the two gonadotrophins may indeed be
produced by separate p i t u i t a r y c e l l types; however, techniques other than immunocytochemistry may have t o be employed i n order t o verffy t h i s . ACKNOWLEDGMENTS The author wishes t o thank Drs. L . Reichert and D.
Ward f o r providing us with t h e hormone preparations, Drs. E. G . Rennels,
M. Shiino and P . Bowie f o r t h e i r advice during t h e course of t h i s invest i g a t i o n , and Miss P. Cisneros and Mrs. M. C a s t i l l e j a f o r t h e i r technical assistance. LITERATURE CITED Baker, B. L . , J . G. Pierceand J . S. Cornell 1972 The u t i l i t y of antiserums t o subunits of TSH and LH f o r immunochemical staining of the r a t hypophysis. h. J . Anat., 135: 251-268. Barrnett, R. J . , E. J. Ladman, N . J . McAllaster and E . R. Siperstein 1956 The localization of glycoprotein hormones i n the a n t e r i o r p i t u i t a r y glands of r a t s investigated by d i f f e r e n t i a l protein s o l u b i l i t i e s , histological s t a i n s and bio-assays. Endocrinology, 59: 398-418. Etreby, M. F., K.-D. Richter and P. Gunzel 1972 Histological and histochemical d i f f e r e n t i a t i o n of the glandular c e l l s of the a n t e r i o r p i t u i t a r y i n various experimental animals. In: Toxicology: Review and Prospect. I n t . Congr. Series No. 219. W . A . M. Duncan, ed. Excerpta Medica, Amsterdam, pp. 270-281. ldebrand, J . E., E. G. Rennels and J. C. Finerty 1957 Gonadotrophic c e l l s of t h e r a t hypophysis and t h e i r r e l a t i o n t o hormone production. Z. Zellforsch. Mikrosk. Anat., 46: 400-411. Kurosumi, K. 1968 Functional c l a s s i f i c a t i o n of c e l l types of t h e a n t e r i o r p i t u i t a r y gland accomplished by electron microscopy. Arch. h i s t o l . j a p . , 29: 329-362. Moriarty, G. C. 1975 Subcellular l o c a l i z a t i o n of l u t e i n i z i n g hormone i n t h e r a t a n t e r i o r p i t u i t a r y : a sex difference in the morphology a n d cytochemistry of LH gonadotrophs. Anat. Rec., 181: 430 (Abstract). Nakane, P. K. 1970 Classifications of a n t e r i o r p i t u i t a r y cell types w i t h imunoenzyme histochemistry. J . Histochem. Cytochem., 18: 9-20. Petrusz, P. 1974 Demonstration of gonadotropin binding s i t e s in the r a t ovary by an immunoglobulin-enzyme bridge method. Europ. J . Obstet. Gynecol. Reprod. Biol. ( S u p p l . ) , 4: S3-S9. Phifer, R. F., A . R. Midgley and S. S. Spicer 1973 Immunohistologic and h i s t o l o g i c evidence t h a t f o l l i c l e - s t i m u l a t i n g hormone and l u t e i n i z i n g hormone a r e present in the same c e l l type i n the human pars d i s t a l i s . J . Clin. Endocrinol. Metab., 36: 125-141. 384
Purves, H . D . , and W. H . Griesbach 1954 The s i t e of f o l l i c l e s t i m u l a t i n g and l u t e i n i z i n g hormone production i n the r a t p i t u i t a r y . Endocrinology, 55: 785-793. Purves, H . D . , and W. H. Griesbach 1955 Changes i n the gonadotrophs of the r a t p i t u i t a r y a f t e r gonadectomy. Endocrinology, 56: 374-386. Rennels, E. G. 1957 Two t i n c t o r i a l types of gonadotrophic c e l l s i n t h e r a t hypophysis. Z. Zellforsch. Mikrosk. Anat., 45: 464-471. Rennels, E. G. 1963 Gonadotrophic c e l l s o f t h e r a t hypophysis. In: Cytologie de 1 'Adenohyophyse. J . Benoit and C. DeLage, eds. C. N . R. S. , P a r i s , pp. 201-213. Schafer, S. J . , and W. H. McShan 1974 Radioimnunoassay d e t e r minations o f gonadotrophic hormone content i n d i f f e r e n t regions o f male and female r a t adenohypophyses. Proc. SOC. Exp. Biol. Med., 146: 546-548. Shiino, M., A. Arimura, A. V. Schally and E . G. Rennels 1972 Ultras t r u c t u r a l observations of granule extrusion from r a t a n t e r i o r p i t u i t a r y c e l l s a f t e r i n j e c t i o n of LH-releasing hormone. Z. Zellforsch. Mikrosk. Anat. , 128: 152-161. S h i i n o , M. , and E . G. Rennels 1968 Immunohistological observations of gonadotrophs i n the a n t e r i o r p i t u i t a r y gland of t h e r a t . Jap. J . Zootech. S c i . , 12: 505-510. Yoshimura, F., and K. Harumiya 1965 Electron microscopy of the a n t e r i o r lobe of the p i t u i t a r y i n normal and c a s t r a t e d rats. Endocrinol. Japan. , 12: 119-151. RE FE REN CE 1 Supported by NIH General Research Support Grant RR05654.
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