Cell Tissue Res. 197, 501-514 (1979)
Cell and Tissue Research 9 by Springer-Verlag 1979
lmmunocytochemical Localisation of Prolactin and Growth Hormone in the Perinatal Sheep Pituitary A Morphological and Quantitative Study D.M. Parry, I.C. McMillen, J.S. Robinson, and G.D. Thorburn* Nuffield Department of Obstetrics and Gynaecology,John RadcliffeHospital, Headington,Oxford, U.K.
Summary. The peroxidase anti-peroxidase immunocytochemical staining technique has been used to identify prolactin and growth hormone cells in pituitaries from fetal and neonatal sheep. The size of the secretory granules in these cell types has been measured using the image analysing computer Quantimet 720. The area size distributions of the fetal prolactin and growth hormone granules were compared with those in the neonate and the adult. It appears that the gestational age of the fetus may influence the size range of prolactin secretory granules. Key words: Prolactin cells - Growth hormone cells - Secretory granules - Size range (quantitative analysis) - Perinatal sheep pituitary.
As yet, there have been few ultrastructural immunocytochemical studies of prolactin and growth hormone (GH) cells except in the rat (Nakane, 1970, 1975; Parsons and Erlandson, 1974), the guinea-pig (Beauvillain et al., 1977), the cow (Dacheux and Dubois, 1976) and the sheep pituitaries (Parry et al., 1978). The only application of systematic quantitative analysis to the prolactin and GH secretory granules has been reported in the sheep (Parry et al., 1978). These authors found that there was a large overlap in the size ranges of the prolactin and GH granules and that it was impossible to distinguish the two cell types on purely morphological grounds. The combination of immunocytochemical staining techniques (peroxidase anti-peroxidase, PAP) and quantitative image analysis has now been applied to the prolactin and GH granules in the fetal sheep pituitary at different stages of gestation and to pituitaries collected from newborn lambs. This allows a Dr. DilysM. Parry, NuffieldDepartmentofObstetricsand Gynaecology,John Radcliffe Hospital, Universityof Oxford, Headington,Oxford, OX3 9DU, England * Present address: Universityof Queensland,Dept. of Physiology,St. Lucia, Brisbane, Queensland 4067, Australia S e n d offprint requests to:
0302-766X/79/0197/0501/$02.80
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c o m p a r i s o n o f t h e s e h o r m o n e s e c r e t o r y g r a n u l e s w i t h t h o s e o f t h e a d u l t ( P a r r y et al., 1978).
Materials and Methods 1. Preparation o f Tissues Pituitaries (7) were collected from both male and female sheep fetuses between 115 days and 144 days gestation and from two newborn lambs on the first day and tenth week after birth. The pituitaries were removed as rapidly as possible after death and small pieces from one half of each fixed in 2 % glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for either 30 min or 2 h at 4 ~C. After overnight washing in buffer, some of the tissue was post-fixed for 30 rain in 1% osmium tetroxide and then alcohol-dehydrated and embedded in Araldite, whilst the remainder was dehydrated and embedded without post-fixative. The second half of each pituitary was fixed in Bouin's fluid and embedded in paraffin for light microscopy.
2. Immunocytochemical Method Reagents: (1) Rabbit anti-ovine prolactin I. (2) Rabbit anti-ovine GH 2.
a) Adsorbed Antisera Adsorption of anti-prolactin with prolactin: 1 ml of 0.04 M phosphate buffer containing 10 ~tg prolactin (NIH P S 11) was added to 1 ml of 1 : 50,000 dilution of prolactin antiserum. The tube was incubated at 37~C for 1 h and then left overnight at 4 ~C. This gives an adsorbed prolactin antiserum at a final dilution of 1 : 100,000. Adsorption of anti-GH with G H : I ml of 0.04M phosphate buffer containing 0.71xg of GH (prepared by the method of Wallace and Ferguson (1963)) was added to 1 ml of 1 : 50,000 dilution of GH antiserum. The procedure for adsorption of GH was the same as that described above for prolactin.
Normal Sheep Serum. Prepared from clotted blood in this laboratory. Sheep Anti-Rabbit Serum. Supplied by Dr. G. Jenkin. Peroxidase-AntioPeroxidase Complex (PAP). Obtained from Miles Laboratory Ltd., Slough, England. Diaminobenzidine (DAB). Obtained from Sigma Co.
b) Electron Microscopy The staining methods used are modifications of the methods of Petrali et al. (1974). All solutions were diluted with 0.05 M phosphate buffer containing 2.5 mg bovine serum albumin (Sigma Co.)/ml. Sections were incubated for 48 h at 4~ in antisera at varying dilutions (1 : 100-1 : 100,000).
1 Kindly supplied by Dr. G. Jenkin, University of Queensland, Department of Physiology, St. Lucia, Brisbane, Queensland 4067, Australia z Kindly supplied by Dr. J.M. Bassett, Nuffield Institute for Medical Research, Headley Way, Headington, Oxford
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c) Light Microscopy Wax sections were cut (7 ~tm) and placed on Teflon coated Multispot slides (obtained from C.A. Hendley &Co., Bucldaurst Hill, Essex). The staining methods used were adapted from Burns (1975) and Petrusz et al. (1975). Sections were incubated in a moisture chamber at 4~ for 24h.
d) Controls For both light and electron microscopy, specificity of staining was tested by substitution of the following solutions for the antiserum: (1) Normal rabbit serum. (2) Anti-prolactin adsorbed with prolactin. (3) Anti-GH adsorbed with GH. (4) Anti-prolactin adsorbed with GH. (5) Anti-GH adsorbed with prolactin. Additional controls were set up omitting sheep anti-rabbit IgG, PAP or DAB.
e) Quantitative Analysis (1) The following procedure was used to assess the number of secretory granules p e r [tm 2 in the prolactin and GH ceils. Random micrographs ofimmunocytochemicallystained granules were taken and the area of the cytoplasm in the total samples estimated by random point analysis. The total number of granules in the sample was counted and the results expressed as granules per pm 2 of cytoplasm. The sample contained a minimum of 1,000 granules. (2) The image analysing computer, Quantimet 720, was used to measure the area of the prolactin and GH secretory granules. Tracings were made of stained granules from electron micrographs at a print magnification of 21,000. For each cell type in each pituitary 1,000--2,000 granules were traced. Microscope magnification was checked by measurement of a diffraction grating replica photographed at the same magnification. The tracings were placed in turn in a wall holder in front of the Vidicon Scanner of the image analysingcomputer which was programmed to measure the area of each traced granule. The programme used incorporated 20 size bins, each with a width of 0.02 I~m and the percentage of the total number of granules falling in each bin was presented on a teletype print-out. From the mean areas the range of granule diameters was calculated, making the assumption that the granules were spherical.
Results Prolactin Cells T h e o p t i m a l d i l u t i o n o f a n t i - p r o l a c t i n for i m m u n o c y t o c h e m i c a l s t a i n i n g was 1 : i 0 , 0 0 0 for light m i c r o s c o p y a n d 1 : 5 0 , 0 0 0 for e l e c t r o n m i c r o s c o p y . N o positive s t a i n i n g was o b s e r v e d w h e n e i t h e r n o r m a l r a b b i t s e r u m o r a n t i p r o l a c t i n a d s o r b e d with p r o l a c t i n were s u b s t i t u t e d for a n t i s e r u m a l o n e (Fig. 1). T h e p r o l a c t i n cells were s p a r s e l y d i s t r i b u t e d , e i t h e r s i n g l y o r i n s m a l l clusters t h r o u g h o u t the fetal a n d n e o n a t a l a n t e r i o r p i t u i t a r y g l a n d . A l t h o u g h difficult to assess a c c u r a t e l y , t h e r e a p p e a r e d to be fewer p r o l a c t i n cells i n t h e fetal p i t u i t a r y t h a n in the n e o n a t a l p i t u i t a r y (Fig. 2 a , b). T h e m o r p h o l o g i c a l a p p e a r a n c e o f the fetal p r o l a c t i n cells a n d o f the o t h e r fetal p i t u i t a r y cell types was c h a r a c t e r i s t i c o f cells v e r y actively e n g a g e d in p r o t e i n synthesis. T h e r e was a b u n d a n t r o u g h s u r f a c e d e n d o p l a s m i c r e t i c u l u m in the fetal
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Fig. l. Sectionshowingno immunocytochemicalstainingin the cytoplasmof fetal pituitary cellsat 118 days of gestation when normal rabbit serum was substituted for anti-prolactin, x 400 pituitary cells of all ages studied and the Golgi apparatus was prominent with many associated microvesicles. The prolactin granules from pituitaries collected between 115 days and 128 days gestation (Fig. 3) appeared smaller and not as densely packed as those observed in the adult pituitaries. However, the protactin granules in the pituitaries collected at 144 days gestation and from the two newborn lambs were larger than those observed in the pituitaries collected earlier in gestation (Fig. 4). The prolactin secretory granules in both the fetal and newborn lamb showed no polymorphy.
Quantitative Analysis of the Prolactin Secretory Granules in the Fetal and Neonatal Pituitaries Using the image analysing computer, the size range of the prolactin granules was measured in seven fetal pituitaries and two newborn lambs. The area size distribution histograms for 1,000-3,000 granules from each pituitary are shown in Fig. 5. The histograms of the pituitaries of similar gestational ages overlapped and have been grouped accordingly. The range of diameter of the granules, the calculated mean areas ( + S.D.) and the percentage of granules with an area greater than 0.1 ~am2 is presented in Table 1. There is a considerable overlap in the range of areas of the granules measured in the perinatal pituitaries but a notably greater proportion of larger granules in the 144 day fetal pituitary and in the neonates than in the pituitaries collected earlier in gestation. Correspondingly, the mean areas of the prolactin granules in these older pituitaries were higher (0.10-0.13 p-m 2) than in the pituitaries removed between 115 days and 128 days gestation (0.02-0.07 p.m2). The proportion of prolactin per p.m 2 of cytoplasm was also larger in the older pituitaries (see Table 3).
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Fig. 2a and b. Immunocytochemical localization of prolactin cells in a fetal sheep pituitary (118 days) and b newborn lamb pituitary. There is a dark staining reaction product over the cytoplasm of the prolacfin cells which are markedly larger in the newborn pituitary, x 400
GH Cells in the Pituitary of the Fetal and Neonatal Sheep The o p t i m a l d i l u t i o n o f the antisera for the identification o f G H cells in the fetal a n d n e o n a t a l pituitaries was 1 : 10,000 for light microscopy a n d 1 : 100,000 for electron microscopy. N o positive s t a i n i n g was observed when n o r m a l s e r u m was s u b s t i t u t e d
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Fig. 3. Immunocytochemical localization of prolactin cells in glutaraldehyde-fixed fetal sheep pituitary (119 days). Prolactin cells (P) stand out in contrast to the unstained cells. (Insert: higher magnification to show DAB reaction product over the granules) • 3460
Fig. 4. Immunocytochemical localization of prolactin cells in glutaraldehyde fixed fetal sheep pituitary (144 days). Prolactin cell (P). The section was counterstained with uranyl acid/lead citrate. • 3460
Prolactin and Growth Hormone Cells: Perinatal Sheep Pituitary
50
5O
40 e 30 w E 20 ~
~
10
~' "6
0
115day 119doy 121day
40 30
I0~ __01
14&day
0.04 0.08 0.12 016 0,20 0.2L
30
20
20
10
10
0
115 day 128 day 128day
20
! 00z, 0.08 0.12 0.16 0,20 0.2~
~ 30
507
New Born 1 day p.p, 10weeks p.p.
0 0.04 D.08 0,12 0.16 0.20 0.24 0.28 0,32 0.04 0.08 0.12 0.16 0.20 0.24 028 0.32 Areo of granules(/,cm 2)
Fig. 5. Histogramto show area sizedistribution of prolactin granulesin nine perinatal ovinepituitaries. There is a greater proportion of larger granules in the 144 day fetal and neonatal pituitaries for the anti-GH alone and adsorption of the anti-GH with prolactin did not affect the intensity of the immunological staining reaction. There appeared to be more G H cells present in the sections of fetal and neonatal pituitaries than prolactin cell types (Fig. 6 a, b), although in this case the numbers of G H cells was similar in both. The cells were found in large dusters and distributed uniformly throughout the anterior lobe. The G H cells were morphologically similar to the prolactin cells with abundant rough surfaced endoplasmic reticulum and active Golgi apparatus and were packed with densely staining large secretory granules (Figs. 7, 8).
Quantitative Analysis of the Growth Hormone Granules in Fetal and Neonatal Pituitaries Using the Quantimet computer, the area size distributions of the G H granules were measured in 5 fetal pituitaries and 2 newborn lambs and the histograms are presented in Fig. 9. It can be seen that the size range of the G H granules in all the pituitaries collected was very similar, although the neonatal pituitaries had consistently higher mean granule areas than those in the fetus (Table 2). The proportion of granules measuring more than 0.1 ~tm2 varied between different animals and the older pituitaries had a consistently higher proportion of larger granules.
Comparison of GH and Prolactin Granules in the Fetal and Neonatal Pituitaries The area size distributions for prolactin and G H granules in the fetal sheep pituitaries almost completely overlap. Before 144 days gestation the fetal prolactin
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Fig. 6a and b. Immunocytochemical localization of GH cells in a fetal sheep pituitary (118 days) and b newborn fetal sheep pituitary, a) • 240; b) • 400
granules range from 113-541 n m in diameter c o m p a r e d to a range o f 113-564 nm for the growth h o r m o n e granules. However, there was a greater p r o p o r t i o n o f larger G H granules ( > 0.10 ktm 2) than prolactin granules in these pituitaries (Tables 1 and 2). A t 144 days gestation and in the n e w b o r n lamb, the size range and mean areas o f the prolactin and G H granules were virtually indistinguishable. There was
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Fig. 7. Immunocytochemical localization of GH cells in a fetal sheep pituitary (115 days). There is a dense reaction product over the granules in the G H cells (G). x 3460
Fig. 8. Immunocytochemical localization of G H cells in a fetal sheep pituitary (144 days). Dense staining reaction product can be seen in three GH (G) cells. The section was counterstained with uranyl acetate/lead citrate, x 3460
B 3O
t
40
115 day 119 day
30
E 2O
20
-5 10
10 0
o
0.04 0.08 0.12 0.16 0.20 0.24
"6
0.04 0.08 0.12 0.16 0.20 0.24
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30
30
20
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-
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1,0
-
-
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0.04 0.08 0.12 0.I6 0.20 0.24 0.04 0.08 0.12 0.16 0.20 0.2l, Area of granules(/zm 2)
Fig.9. Histogram to show area size distributions of GH granules in seven neonatal ovine pituitaries
1. Results from a Quantimet analysis of the prolactin secretory granules in seven fetal and two neonatal pituitaries Table
Age of fetus/lamb
No. of granules measured
Range of diam. Mean area of granules (nm) + S.D. (~tm2)
70 of granules > 0.10 ~tm2
115 days 119 days 121 days 125 days 128 days 128 days 144 days Newborn I day post partum Newborn 10 weeks post partum
1031 2651 1914 1024 2358 2250 3002
113-491 113-407 113-407 113-437 113-407 113-541 113-686
0.07+ 0.02+ 0.03 + 0.04+ 0.02 + 0.05 + 0.12 +
16% 0.1 70 0.5 % 1.27oo 0.25 % 4.5 % 59.0 %
1871
113-628
0.10 + 0.04
44.0 %
1450
113-704
0.13 + 0.06
59.0 %
0.003 0.01 0.02 0.02 0.02 0.03 0.05
2. Results from a Quantimet analysis of the growth hormone secretory granules in five fetal and two neonatal pituitaries
Table
Age of fetus/lamb
No. of granules measured
Range of diam. Mean area of granules (nm) + S.D. (lam2)
70 of granules > 0.10 ~tm2
115 days 119 days 121 days 125 days 144 days Newborn 1 day post partum Newborn 10 weeks post partum
1465 2073 1103 2314 3143
113-564 113-607 113-517 113-465 113-586
0.072+ 0.089 + 0.056+ 0.054+ 0.067+
14.5% 30.0 % 4.3% 3.0% 11.0%
1823
113-564
0.089 + 0.033
31.9 %
1240
113-586
0.099 + 0.034
39.5 %
0.029 0.033 0.023 0.022 0.029
Prolactin and Growth Hormone Cells: Perinatal Sheep Pituitary
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Table 3. Estimated mean area of protein hormone per ~tmz of cytoplasm
Age of fetus or lamb
Prolactin per ~tm2
Growth hormone per lamz
115 days 119 days 121 days 125 days 128 days
0.167 0.110 0.144 0.209 0.107 0.142 0.260 0.350 0.313
0.255 0.408 0.228 0.282 -* - * 0.328 0.415 0.413
144 Newborn day 1 Newborn week 10 *
not measured
a consistently larger proportion of protein hormone in the cytoplasm of the fetal and neonatal G H cells, than in the corresponding prolactin cells (Table 3).
Discussion
The morphology of the G H ceils in the fetal pituitary resembled that of the G H cell types in the pituitary of the adult (Parry et al., 1978) and the newborn sheep. The only other ultrastructural immunocytochemical study of somatotrophs in the fetus to date is that o f L i et al. (1978) in the h u m a n fetal pituitary. There authors found that the mean diameter of the G H secretory granules (300 nm - mean of 400 measurements) was the same as that found at 19 weeks gestation. They did note, however, that there was a large variation in the mean secretory granule size from one s o m a t o t r o p h to another. In the present study, the range of diameter of the G H granules varied from 113-465 nm (at 125 days of gestation) to 113-607 nm (at 119 days o f gestation). The mean areas o f the fetal G H granules were similar throughout gestation as was the proportion of G H per ~tm2 of cytoplasm found in each of these pituitaries. The fetal sheep plasma concentrations o f G H are approximately tenfold higher than the concentrations of this hormone in the maternal plasma (Bassett et al., 1970). This is probably a result of high secretion of growth hormone by the fetal pituitary rather than as a result of slower fetal clearance o f G H . This difference in the fetal and adult secretory rate o f G H is not accompanied by any gross ultrastructural difference in the fetal pituitary growth hormone granules. It would appear that by 115 days o f gestation, t h e fetal somatotroph is well differentiated and the area size distribution of the G H granules very closely resembles that in the adult sheep pituitary (Parry et al., 1978). The fetal somatotroph contains proportionately more protein hormone per unit area of cytoplasm than does the fetal prolactin cell. This, coupled with the observation of more G H cells than prolactin cells in the fetal pituitary sections, would imply that the fetal anterior pituitary has a larger store of growth h o r m o n e than o f prolactin. The area size distribution curves determined for the prolactin and G H granules in the same fetal pituitaries had a considerable area of overlap. Both the G H and
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prolactin granules ranged from 113 nm-600 nm in diameter. Alexander et al. (1973) identified GH and prolactin cell types in the fetal sheep pituitary on purely ultrastructural grounds. These authors quoted the diameter of the fetal GH granules as around 290nm and the diameter of the fetal prolactin granules as around 360 nm. Although it would seem likely from the present study that granules of this order of size would be either prolactin or GH it would also seem impossible to distinguish the two cell types without an immunocytochemical stain. It is interesting in the present study that in fetal pituitaries collected between 115 and 128 days of gestation the prolactin cells contained less hormone per unit area of cytoplasm than found in the adult pituitary. The prolactin cells were also sparsely distributed throughout the fetal pituitary sections. This would suggest that the fetal pituitary prolactin store is lower than that of the adult pituitary. There is, however, a close overlap of the area size distribution curves for the prolactin granules in the fetal pituitaries (115-128 days gestation) with the area size distribution curve of the prolactin granules measured in the anestrous ewe. A similar relationship exists between the size distributions of the prolactin granules in the late fetal and neonatal pituitaries with those in the post-partum ewe (Parry et al., 1978). This indicates that the packaging of prolactin into granules in the pituitary cells of the fetus in the last third of gestation is fully developed and comparable to that in the adult sheep pituitary. There was a striking increase in the proportion of larger granules ( > 0.1 p.m 2) in the fetal pituitary taken at 144 days of gestation compared with pituitaries collected earlier in gestation. In late gestation and in the neonatal period, the mean areas of the prolactin granules were larger than those in the early fetal pituitary. The increase in the proportion of prolactin per pm 2 of cytoplasm and granule diameter which occurs in the fetus at 144 days of gestation correlates with the increase in fetal circulating concentrations of prolactin which is a feature of late pregnancy in many species (Kaplan et al., 1976). There does not appear to be such a direct relationship between the growth hormone granule diameter and the secretory rate of this hormone from the pituitary somatotroph. This may be a function of a larger less transient store of growth hormone in the ovine pituitary. It seems that when pituitary prolactin secretion is stimulated there is an overall increase in the cellular production of prolactin which is packaged into larger granules. In an autoradiographic study on the rat pituitary, Farquhar et al. (1978) have described four types of prolactin granule which appeared to represent different stages of the packaging of the hormone into granules. Early storage forms were small and round, intermediate forms were aggregated and typically polymorphic, and the more mature granules were round or ovoid. It may be that the large round granules observed in the sheep pituitary are a result of aggregation of smaller hormone granules. However, it is interesting that the irregular granules such as those seen in the rat pituitary were never observed in the ovine prolactin cells. These might be expected to occur as intermediate forms between the small and large spherical granules. Another explanation of the larger ovine prolactin granules may be that they represent the packaging of a different form of prolactin molecule. The existence of multiple forms of prolactin has been demonstrated in the human pituitary gland and serum (Hwang et al., 1973; Suh and Frantz, 1974). It would be necessary to chromatograph serum from adult and fetal sheep to determine whether
Prolactin and Growth Hormone Cells: Perinatal Sheep Pituitary
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there are different molecular forms of prolactin present similar to those found in the human. Whilst prolactin granules in the pituitary are identified with antisera which will recognise several forms of the molecule, it will be impossible to determine whether these different forms are packaged separately. In summary, this is the first application of systematic quantitation to pituitary hormone granules identified with a specific immunocytochemical stain in the fetal sheep. It appears that the area size distribution curves for both prolactin and GH granules considerably overlap. There are, however, differences in the size distribution of the prolactin granules in different fetal pituitaries which may be dependent on the gestational age of the animal.
Acknowledgements. We wish to thank Drs J.M. Bassett and G. Jenkin for the gift of antisera. We are indebted to Elaine Johnston, Eileen Stanley and Jane Kingston for their skilled technical assistance and to Dr. S. Bradbury and the Department of Human Anatomy, Oxford University for the use of the Quantimet 720 and the computer programme. ICMc is an MRC postgraduate student.
References Alexander, D.P., Britton, H.G., Cameron, E., Nixon, C.L., Foster, D.A.: Adenohypophysis of foetal sheep: correlation of ultrastructure with functional activity. J. Physiol. (Lond.) 230, 10-13P (1973) Bassett, J.M., Thorburn, G.D., Wallace, A.L.C.: The plasma growth hormone concentration of the foetal lamb. J. Endocrinol. 48, 251-263 (1970) Beauvillain, J.C., Mazzuca, M., Dubois, M.P.: The prolactin and growth hormone producing cells of the guinea-pig pituitary. Cell. Tissue Res. 184, 343-358 (1977) Bums, J.: Background staining and sensitivity of the unlabelled antibody-enzyme (PAP) method. Comparison with the peroxidase labelled antibody sandwich method using formalin fixed paraffin embedded material. Histochemistry 43, 291-294 (1975) Dacheux, F., Dubois, M.P.: Ultrastructural localisation of prolactin, growth hormone and luteinising hormone by immunocytochemical techniques in the bovine pituitary. Cell. Tissue Res. 174, 245-260 (1976) Farquhar, M.G., Reid, J.J., Daniell, L.W.: Intracellular transport and packaging of prolactin. A quantitative electron microscope autoradiographic study of mammotrophs dissociated from rat pituitaries. Endocrinology 102, 296-311 (1978) Hwang, P., Robertson, M., Guyda, H., Friesen, H.: The purification of human prolactin from frozen pituitary glands. J. Clin. Endocr. Metab. 36, 110-118 (1973) Kaplan, S.L., Grumbach, M.M., Aubert, M.L.: The ontogenesis of pituitary hormones and hypothalamic factors in the human fetus: maturation of central nervous system regulation of anterior pituitary function. Rec. Prog. Horm. Res. 32, 161-243 (1976) Li, J.Y., Dubois, M.P., Dubois, P.M.: Somatotrophs in the human fetal anterior pituitary. Cell Tissue Res. 181, 545-552 (1978) Nakane, P.K.: Classification of anterior pituitary cell types with immunoenzyme histochemistry. J. Histochem. Cytochem. 18, 9-20 (1970) Nakane, P.K.: Identification of anterior pituitary cells by immunoelectron microscopy. In: The Anterior Pituitary. (A. Tixier-Vidal and M.G. Farquhar, eds.) New York: Academic Press Parry, D.M., McMillen, I.C., Willcox, D.L.: Immunocytochemical localisation of prolactin and growth hormone in the ovine pituitary. A morphological and quantitative study. Cell Tissue Res. (in press) (1978) Parsons, J.A., Erlandsen, S.L.: Ultrastructural immunocytochemical localisation of prolactin in rat anterior pituitary by use of the unlabeled antibody enzyme method. J. Histochem. Cytochem. 22, 340-352 (1974)
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Petrali, J.P., Hinton, D.M., Moriarty, G.C., Sternberger, L.A.: The unlabeled antibody enzyme method of immunocytochemistry. Quantitative comparison of sensitivities with and without peroxidaseantiperoxidase complex. J. Histochem. Cytochem. 22, 782-801 (1974) Petrusz, P., DiMeo, P., Ordronneau, P., Weaver, C., Keefer, D.A.: Improved immunoglobulin-enzyme bridge method for light microscopic demonstration of hormone-containing cells of the rat adenohypophysis. Histochemistry 46, 9-26 (1975) Suh, H.K., Frantz, A.G.: Size heterogeneity of human prolactin in plasma and pituitary extracts. J. Clin. Endocr. Metab. 39, 328-935 (1974) Wallace, A.L.C., Ferguson, K.A.: The preparation of sheep growth hormone. J. Endocrinol. 26, 254263 (1963) Accepted December 9, 1978
Cell and Tissue Research 9 by Springer-Verlag 1979
lmmunocytochemical Localisation of Prolactin and Growth Hormone in the Perinatal Sheep Pituitary A Morphological and Quantitative Study D.M. Parry, I.C. McMillen, J.S. Robinson, and G.D. Thorburn* Nuffield Department of Obstetrics and Gynaecology,John RadcliffeHospital, Headington,Oxford, U.K.
Summary. The peroxidase anti-peroxidase immunocytochemical staining technique has been used to identify prolactin and growth hormone cells in pituitaries from fetal and neonatal sheep. The size of the secretory granules in these cell types has been measured using the image analysing computer Quantimet 720. The area size distributions of the fetal prolactin and growth hormone granules were compared with those in the neonate and the adult. It appears that the gestational age of the fetus may influence the size range of prolactin secretory granules. Key words: Prolactin cells - Growth hormone cells - Secretory granules - Size range (quantitative analysis) - Perinatal sheep pituitary.
As yet, there have been few ultrastructural immunocytochemical studies of prolactin and growth hormone (GH) cells except in the rat (Nakane, 1970, 1975; Parsons and Erlandson, 1974), the guinea-pig (Beauvillain et al., 1977), the cow (Dacheux and Dubois, 1976) and the sheep pituitaries (Parry et al., 1978). The only application of systematic quantitative analysis to the prolactin and GH secretory granules has been reported in the sheep (Parry et al., 1978). These authors found that there was a large overlap in the size ranges of the prolactin and GH granules and that it was impossible to distinguish the two cell types on purely morphological grounds. The combination of immunocytochemical staining techniques (peroxidase anti-peroxidase, PAP) and quantitative image analysis has now been applied to the prolactin and GH granules in the fetal sheep pituitary at different stages of gestation and to pituitaries collected from newborn lambs. This allows a Dr. DilysM. Parry, NuffieldDepartmentofObstetricsand Gynaecology,John Radcliffe Hospital, Universityof Oxford, Headington,Oxford, OX3 9DU, England * Present address: Universityof Queensland,Dept. of Physiology,St. Lucia, Brisbane, Queensland 4067, Australia S e n d offprint requests to:
0302-766X/79/0197/0501/$02.80
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c o m p a r i s o n o f t h e s e h o r m o n e s e c r e t o r y g r a n u l e s w i t h t h o s e o f t h e a d u l t ( P a r r y et al., 1978).
Materials and Methods 1. Preparation o f Tissues Pituitaries (7) were collected from both male and female sheep fetuses between 115 days and 144 days gestation and from two newborn lambs on the first day and tenth week after birth. The pituitaries were removed as rapidly as possible after death and small pieces from one half of each fixed in 2 % glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for either 30 min or 2 h at 4 ~C. After overnight washing in buffer, some of the tissue was post-fixed for 30 rain in 1% osmium tetroxide and then alcohol-dehydrated and embedded in Araldite, whilst the remainder was dehydrated and embedded without post-fixative. The second half of each pituitary was fixed in Bouin's fluid and embedded in paraffin for light microscopy.
2. Immunocytochemical Method Reagents: (1) Rabbit anti-ovine prolactin I. (2) Rabbit anti-ovine GH 2.
a) Adsorbed Antisera Adsorption of anti-prolactin with prolactin: 1 ml of 0.04 M phosphate buffer containing 10 ~tg prolactin (NIH P S 11) was added to 1 ml of 1 : 50,000 dilution of prolactin antiserum. The tube was incubated at 37~C for 1 h and then left overnight at 4 ~C. This gives an adsorbed prolactin antiserum at a final dilution of 1 : 100,000. Adsorption of anti-GH with G H : I ml of 0.04M phosphate buffer containing 0.71xg of GH (prepared by the method of Wallace and Ferguson (1963)) was added to 1 ml of 1 : 50,000 dilution of GH antiserum. The procedure for adsorption of GH was the same as that described above for prolactin.
Normal Sheep Serum. Prepared from clotted blood in this laboratory. Sheep Anti-Rabbit Serum. Supplied by Dr. G. Jenkin. Peroxidase-AntioPeroxidase Complex (PAP). Obtained from Miles Laboratory Ltd., Slough, England. Diaminobenzidine (DAB). Obtained from Sigma Co.
b) Electron Microscopy The staining methods used are modifications of the methods of Petrali et al. (1974). All solutions were diluted with 0.05 M phosphate buffer containing 2.5 mg bovine serum albumin (Sigma Co.)/ml. Sections were incubated for 48 h at 4~ in antisera at varying dilutions (1 : 100-1 : 100,000).
1 Kindly supplied by Dr. G. Jenkin, University of Queensland, Department of Physiology, St. Lucia, Brisbane, Queensland 4067, Australia z Kindly supplied by Dr. J.M. Bassett, Nuffield Institute for Medical Research, Headley Way, Headington, Oxford
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c) Light Microscopy Wax sections were cut (7 ~tm) and placed on Teflon coated Multispot slides (obtained from C.A. Hendley &Co., Bucldaurst Hill, Essex). The staining methods used were adapted from Burns (1975) and Petrusz et al. (1975). Sections were incubated in a moisture chamber at 4~ for 24h.
d) Controls For both light and electron microscopy, specificity of staining was tested by substitution of the following solutions for the antiserum: (1) Normal rabbit serum. (2) Anti-prolactin adsorbed with prolactin. (3) Anti-GH adsorbed with GH. (4) Anti-prolactin adsorbed with GH. (5) Anti-GH adsorbed with prolactin. Additional controls were set up omitting sheep anti-rabbit IgG, PAP or DAB.
e) Quantitative Analysis (1) The following procedure was used to assess the number of secretory granules p e r [tm 2 in the prolactin and GH ceils. Random micrographs ofimmunocytochemicallystained granules were taken and the area of the cytoplasm in the total samples estimated by random point analysis. The total number of granules in the sample was counted and the results expressed as granules per pm 2 of cytoplasm. The sample contained a minimum of 1,000 granules. (2) The image analysing computer, Quantimet 720, was used to measure the area of the prolactin and GH secretory granules. Tracings were made of stained granules from electron micrographs at a print magnification of 21,000. For each cell type in each pituitary 1,000--2,000 granules were traced. Microscope magnification was checked by measurement of a diffraction grating replica photographed at the same magnification. The tracings were placed in turn in a wall holder in front of the Vidicon Scanner of the image analysingcomputer which was programmed to measure the area of each traced granule. The programme used incorporated 20 size bins, each with a width of 0.02 I~m and the percentage of the total number of granules falling in each bin was presented on a teletype print-out. From the mean areas the range of granule diameters was calculated, making the assumption that the granules were spherical.
Results Prolactin Cells T h e o p t i m a l d i l u t i o n o f a n t i - p r o l a c t i n for i m m u n o c y t o c h e m i c a l s t a i n i n g was 1 : i 0 , 0 0 0 for light m i c r o s c o p y a n d 1 : 5 0 , 0 0 0 for e l e c t r o n m i c r o s c o p y . N o positive s t a i n i n g was o b s e r v e d w h e n e i t h e r n o r m a l r a b b i t s e r u m o r a n t i p r o l a c t i n a d s o r b e d with p r o l a c t i n were s u b s t i t u t e d for a n t i s e r u m a l o n e (Fig. 1). T h e p r o l a c t i n cells were s p a r s e l y d i s t r i b u t e d , e i t h e r s i n g l y o r i n s m a l l clusters t h r o u g h o u t the fetal a n d n e o n a t a l a n t e r i o r p i t u i t a r y g l a n d . A l t h o u g h difficult to assess a c c u r a t e l y , t h e r e a p p e a r e d to be fewer p r o l a c t i n cells i n t h e fetal p i t u i t a r y t h a n in the n e o n a t a l p i t u i t a r y (Fig. 2 a , b). T h e m o r p h o l o g i c a l a p p e a r a n c e o f the fetal p r o l a c t i n cells a n d o f the o t h e r fetal p i t u i t a r y cell types was c h a r a c t e r i s t i c o f cells v e r y actively e n g a g e d in p r o t e i n synthesis. T h e r e was a b u n d a n t r o u g h s u r f a c e d e n d o p l a s m i c r e t i c u l u m in the fetal
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Fig. l. Sectionshowingno immunocytochemicalstainingin the cytoplasmof fetal pituitary cellsat 118 days of gestation when normal rabbit serum was substituted for anti-prolactin, x 400 pituitary cells of all ages studied and the Golgi apparatus was prominent with many associated microvesicles. The prolactin granules from pituitaries collected between 115 days and 128 days gestation (Fig. 3) appeared smaller and not as densely packed as those observed in the adult pituitaries. However, the protactin granules in the pituitaries collected at 144 days gestation and from the two newborn lambs were larger than those observed in the pituitaries collected earlier in gestation (Fig. 4). The prolactin secretory granules in both the fetal and newborn lamb showed no polymorphy.
Quantitative Analysis of the Prolactin Secretory Granules in the Fetal and Neonatal Pituitaries Using the image analysing computer, the size range of the prolactin granules was measured in seven fetal pituitaries and two newborn lambs. The area size distribution histograms for 1,000-3,000 granules from each pituitary are shown in Fig. 5. The histograms of the pituitaries of similar gestational ages overlapped and have been grouped accordingly. The range of diameter of the granules, the calculated mean areas ( + S.D.) and the percentage of granules with an area greater than 0.1 ~am2 is presented in Table 1. There is a considerable overlap in the range of areas of the granules measured in the perinatal pituitaries but a notably greater proportion of larger granules in the 144 day fetal pituitary and in the neonates than in the pituitaries collected earlier in gestation. Correspondingly, the mean areas of the prolactin granules in these older pituitaries were higher (0.10-0.13 p-m 2) than in the pituitaries removed between 115 days and 128 days gestation (0.02-0.07 p.m2). The proportion of prolactin per p.m 2 of cytoplasm was also larger in the older pituitaries (see Table 3).
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Fig. 2a and b. Immunocytochemical localization of prolactin cells in a fetal sheep pituitary (118 days) and b newborn lamb pituitary. There is a dark staining reaction product over the cytoplasm of the prolacfin cells which are markedly larger in the newborn pituitary, x 400
GH Cells in the Pituitary of the Fetal and Neonatal Sheep The o p t i m a l d i l u t i o n o f the antisera for the identification o f G H cells in the fetal a n d n e o n a t a l pituitaries was 1 : 10,000 for light microscopy a n d 1 : 100,000 for electron microscopy. N o positive s t a i n i n g was observed when n o r m a l s e r u m was s u b s t i t u t e d
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Fig. 3. Immunocytochemical localization of prolactin cells in glutaraldehyde-fixed fetal sheep pituitary (119 days). Prolactin cells (P) stand out in contrast to the unstained cells. (Insert: higher magnification to show DAB reaction product over the granules) • 3460
Fig. 4. Immunocytochemical localization of prolactin cells in glutaraldehyde fixed fetal sheep pituitary (144 days). Prolactin cell (P). The section was counterstained with uranyl acid/lead citrate. • 3460
Prolactin and Growth Hormone Cells: Perinatal Sheep Pituitary
50
5O
40 e 30 w E 20 ~
~
10
~' "6
0
115day 119doy 121day
40 30
I0~ __01
14&day
0.04 0.08 0.12 016 0,20 0.2L
30
20
20
10
10
0
115 day 128 day 128day
20
! 00z, 0.08 0.12 0.16 0,20 0.2~
~ 30
507
New Born 1 day p.p, 10weeks p.p.
0 0.04 D.08 0,12 0.16 0.20 0.24 0.28 0,32 0.04 0.08 0.12 0.16 0.20 0.24 028 0.32 Areo of granules(/,cm 2)
Fig. 5. Histogramto show area sizedistribution of prolactin granulesin nine perinatal ovinepituitaries. There is a greater proportion of larger granules in the 144 day fetal and neonatal pituitaries for the anti-GH alone and adsorption of the anti-GH with prolactin did not affect the intensity of the immunological staining reaction. There appeared to be more G H cells present in the sections of fetal and neonatal pituitaries than prolactin cell types (Fig. 6 a, b), although in this case the numbers of G H cells was similar in both. The cells were found in large dusters and distributed uniformly throughout the anterior lobe. The G H cells were morphologically similar to the prolactin cells with abundant rough surfaced endoplasmic reticulum and active Golgi apparatus and were packed with densely staining large secretory granules (Figs. 7, 8).
Quantitative Analysis of the Growth Hormone Granules in Fetal and Neonatal Pituitaries Using the Quantimet computer, the area size distributions of the G H granules were measured in 5 fetal pituitaries and 2 newborn lambs and the histograms are presented in Fig. 9. It can be seen that the size range of the G H granules in all the pituitaries collected was very similar, although the neonatal pituitaries had consistently higher mean granule areas than those in the fetus (Table 2). The proportion of granules measuring more than 0.1 ~tm2 varied between different animals and the older pituitaries had a consistently higher proportion of larger granules.
Comparison of GH and Prolactin Granules in the Fetal and Neonatal Pituitaries The area size distributions for prolactin and G H granules in the fetal sheep pituitaries almost completely overlap. Before 144 days gestation the fetal prolactin
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Fig. 6a and b. Immunocytochemical localization of GH cells in a fetal sheep pituitary (118 days) and b newborn fetal sheep pituitary, a) • 240; b) • 400
granules range from 113-541 n m in diameter c o m p a r e d to a range o f 113-564 nm for the growth h o r m o n e granules. However, there was a greater p r o p o r t i o n o f larger G H granules ( > 0.10 ktm 2) than prolactin granules in these pituitaries (Tables 1 and 2). A t 144 days gestation and in the n e w b o r n lamb, the size range and mean areas o f the prolactin and G H granules were virtually indistinguishable. There was
Prolactin and Growth Hormone Cells: Perinatal Sheep Pituitary
509
Fig. 7. Immunocytochemical localization of GH cells in a fetal sheep pituitary (115 days). There is a dense reaction product over the granules in the G H cells (G). x 3460
Fig. 8. Immunocytochemical localization of G H cells in a fetal sheep pituitary (144 days). Dense staining reaction product can be seen in three GH (G) cells. The section was counterstained with uranyl acetate/lead citrate, x 3460
B 3O
t
40
115 day 119 day
30
E 2O
20
-5 10
10 0
o
0.04 0.08 0.12 0.16 0.20 0.24
"6
0.04 0.08 0.12 0.16 0.20 0.24
1L4doy c
30
30
20
20
~ 10
10
-
o
"5 ~
New Born
1,0
-
-
0
0.04 0.08 0.12 0.I6 0.20 0.24 0.04 0.08 0.12 0.16 0.20 0.2l, Area of granules(/zm 2)
Fig.9. Histogram to show area size distributions of GH granules in seven neonatal ovine pituitaries
1. Results from a Quantimet analysis of the prolactin secretory granules in seven fetal and two neonatal pituitaries Table
Age of fetus/lamb
No. of granules measured
Range of diam. Mean area of granules (nm) + S.D. (~tm2)
70 of granules > 0.10 ~tm2
115 days 119 days 121 days 125 days 128 days 128 days 144 days Newborn I day post partum Newborn 10 weeks post partum
1031 2651 1914 1024 2358 2250 3002
113-491 113-407 113-407 113-437 113-407 113-541 113-686
0.07+ 0.02+ 0.03 + 0.04+ 0.02 + 0.05 + 0.12 +
16% 0.1 70 0.5 % 1.27oo 0.25 % 4.5 % 59.0 %
1871
113-628
0.10 + 0.04
44.0 %
1450
113-704
0.13 + 0.06
59.0 %
0.003 0.01 0.02 0.02 0.02 0.03 0.05
2. Results from a Quantimet analysis of the growth hormone secretory granules in five fetal and two neonatal pituitaries
Table
Age of fetus/lamb
No. of granules measured
Range of diam. Mean area of granules (nm) + S.D. (lam2)
70 of granules > 0.10 ~tm2
115 days 119 days 121 days 125 days 144 days Newborn 1 day post partum Newborn 10 weeks post partum
1465 2073 1103 2314 3143
113-564 113-607 113-517 113-465 113-586
0.072+ 0.089 + 0.056+ 0.054+ 0.067+
14.5% 30.0 % 4.3% 3.0% 11.0%
1823
113-564
0.089 + 0.033
31.9 %
1240
113-586
0.099 + 0.034
39.5 %
0.029 0.033 0.023 0.022 0.029
Prolactin and Growth Hormone Cells: Perinatal Sheep Pituitary
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Table 3. Estimated mean area of protein hormone per ~tmz of cytoplasm
Age of fetus or lamb
Prolactin per ~tm2
Growth hormone per lamz
115 days 119 days 121 days 125 days 128 days
0.167 0.110 0.144 0.209 0.107 0.142 0.260 0.350 0.313
0.255 0.408 0.228 0.282 -* - * 0.328 0.415 0.413
144 Newborn day 1 Newborn week 10 *
not measured
a consistently larger proportion of protein hormone in the cytoplasm of the fetal and neonatal G H cells, than in the corresponding prolactin cells (Table 3).
Discussion
The morphology of the G H ceils in the fetal pituitary resembled that of the G H cell types in the pituitary of the adult (Parry et al., 1978) and the newborn sheep. The only other ultrastructural immunocytochemical study of somatotrophs in the fetus to date is that o f L i et al. (1978) in the h u m a n fetal pituitary. There authors found that the mean diameter of the G H secretory granules (300 nm - mean of 400 measurements) was the same as that found at 19 weeks gestation. They did note, however, that there was a large variation in the mean secretory granule size from one s o m a t o t r o p h to another. In the present study, the range of diameter of the G H granules varied from 113-465 nm (at 125 days of gestation) to 113-607 nm (at 119 days o f gestation). The mean areas o f the fetal G H granules were similar throughout gestation as was the proportion of G H per ~tm2 of cytoplasm found in each of these pituitaries. The fetal sheep plasma concentrations o f G H are approximately tenfold higher than the concentrations of this hormone in the maternal plasma (Bassett et al., 1970). This is probably a result of high secretion of growth hormone by the fetal pituitary rather than as a result of slower fetal clearance o f G H . This difference in the fetal and adult secretory rate o f G H is not accompanied by any gross ultrastructural difference in the fetal pituitary growth hormone granules. It would appear that by 115 days o f gestation, t h e fetal somatotroph is well differentiated and the area size distribution of the G H granules very closely resembles that in the adult sheep pituitary (Parry et al., 1978). The fetal somatotroph contains proportionately more protein hormone per unit area of cytoplasm than does the fetal prolactin cell. This, coupled with the observation of more G H cells than prolactin cells in the fetal pituitary sections, would imply that the fetal anterior pituitary has a larger store of growth h o r m o n e than o f prolactin. The area size distribution curves determined for the prolactin and G H granules in the same fetal pituitaries had a considerable area of overlap. Both the G H and
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prolactin granules ranged from 113 nm-600 nm in diameter. Alexander et al. (1973) identified GH and prolactin cell types in the fetal sheep pituitary on purely ultrastructural grounds. These authors quoted the diameter of the fetal GH granules as around 290nm and the diameter of the fetal prolactin granules as around 360 nm. Although it would seem likely from the present study that granules of this order of size would be either prolactin or GH it would also seem impossible to distinguish the two cell types without an immunocytochemical stain. It is interesting in the present study that in fetal pituitaries collected between 115 and 128 days of gestation the prolactin cells contained less hormone per unit area of cytoplasm than found in the adult pituitary. The prolactin cells were also sparsely distributed throughout the fetal pituitary sections. This would suggest that the fetal pituitary prolactin store is lower than that of the adult pituitary. There is, however, a close overlap of the area size distribution curves for the prolactin granules in the fetal pituitaries (115-128 days gestation) with the area size distribution curve of the prolactin granules measured in the anestrous ewe. A similar relationship exists between the size distributions of the prolactin granules in the late fetal and neonatal pituitaries with those in the post-partum ewe (Parry et al., 1978). This indicates that the packaging of prolactin into granules in the pituitary cells of the fetus in the last third of gestation is fully developed and comparable to that in the adult sheep pituitary. There was a striking increase in the proportion of larger granules ( > 0.1 p.m 2) in the fetal pituitary taken at 144 days of gestation compared with pituitaries collected earlier in gestation. In late gestation and in the neonatal period, the mean areas of the prolactin granules were larger than those in the early fetal pituitary. The increase in the proportion of prolactin per pm 2 of cytoplasm and granule diameter which occurs in the fetus at 144 days of gestation correlates with the increase in fetal circulating concentrations of prolactin which is a feature of late pregnancy in many species (Kaplan et al., 1976). There does not appear to be such a direct relationship between the growth hormone granule diameter and the secretory rate of this hormone from the pituitary somatotroph. This may be a function of a larger less transient store of growth hormone in the ovine pituitary. It seems that when pituitary prolactin secretion is stimulated there is an overall increase in the cellular production of prolactin which is packaged into larger granules. In an autoradiographic study on the rat pituitary, Farquhar et al. (1978) have described four types of prolactin granule which appeared to represent different stages of the packaging of the hormone into granules. Early storage forms were small and round, intermediate forms were aggregated and typically polymorphic, and the more mature granules were round or ovoid. It may be that the large round granules observed in the sheep pituitary are a result of aggregation of smaller hormone granules. However, it is interesting that the irregular granules such as those seen in the rat pituitary were never observed in the ovine prolactin cells. These might be expected to occur as intermediate forms between the small and large spherical granules. Another explanation of the larger ovine prolactin granules may be that they represent the packaging of a different form of prolactin molecule. The existence of multiple forms of prolactin has been demonstrated in the human pituitary gland and serum (Hwang et al., 1973; Suh and Frantz, 1974). It would be necessary to chromatograph serum from adult and fetal sheep to determine whether
Prolactin and Growth Hormone Cells: Perinatal Sheep Pituitary
513
there are different molecular forms of prolactin present similar to those found in the human. Whilst prolactin granules in the pituitary are identified with antisera which will recognise several forms of the molecule, it will be impossible to determine whether these different forms are packaged separately. In summary, this is the first application of systematic quantitation to pituitary hormone granules identified with a specific immunocytochemical stain in the fetal sheep. It appears that the area size distribution curves for both prolactin and GH granules considerably overlap. There are, however, differences in the size distribution of the prolactin granules in different fetal pituitaries which may be dependent on the gestational age of the animal.
Acknowledgements. We wish to thank Drs J.M. Bassett and G. Jenkin for the gift of antisera. We are indebted to Elaine Johnston, Eileen Stanley and Jane Kingston for their skilled technical assistance and to Dr. S. Bradbury and the Department of Human Anatomy, Oxford University for the use of the Quantimet 720 and the computer programme. ICMc is an MRC postgraduate student.
References Alexander, D.P., Britton, H.G., Cameron, E., Nixon, C.L., Foster, D.A.: Adenohypophysis of foetal sheep: correlation of ultrastructure with functional activity. J. Physiol. (Lond.) 230, 10-13P (1973) Bassett, J.M., Thorburn, G.D., Wallace, A.L.C.: The plasma growth hormone concentration of the foetal lamb. J. Endocrinol. 48, 251-263 (1970) Beauvillain, J.C., Mazzuca, M., Dubois, M.P.: The prolactin and growth hormone producing cells of the guinea-pig pituitary. Cell. Tissue Res. 184, 343-358 (1977) Bums, J.: Background staining and sensitivity of the unlabelled antibody-enzyme (PAP) method. Comparison with the peroxidase labelled antibody sandwich method using formalin fixed paraffin embedded material. Histochemistry 43, 291-294 (1975) Dacheux, F., Dubois, M.P.: Ultrastructural localisation of prolactin, growth hormone and luteinising hormone by immunocytochemical techniques in the bovine pituitary. Cell. Tissue Res. 174, 245-260 (1976) Farquhar, M.G., Reid, J.J., Daniell, L.W.: Intracellular transport and packaging of prolactin. A quantitative electron microscope autoradiographic study of mammotrophs dissociated from rat pituitaries. Endocrinology 102, 296-311 (1978) Hwang, P., Robertson, M., Guyda, H., Friesen, H.: The purification of human prolactin from frozen pituitary glands. J. Clin. Endocr. Metab. 36, 110-118 (1973) Kaplan, S.L., Grumbach, M.M., Aubert, M.L.: The ontogenesis of pituitary hormones and hypothalamic factors in the human fetus: maturation of central nervous system regulation of anterior pituitary function. Rec. Prog. Horm. Res. 32, 161-243 (1976) Li, J.Y., Dubois, M.P., Dubois, P.M.: Somatotrophs in the human fetal anterior pituitary. Cell Tissue Res. 181, 545-552 (1978) Nakane, P.K.: Classification of anterior pituitary cell types with immunoenzyme histochemistry. J. Histochem. Cytochem. 18, 9-20 (1970) Nakane, P.K.: Identification of anterior pituitary cells by immunoelectron microscopy. In: The Anterior Pituitary. (A. Tixier-Vidal and M.G. Farquhar, eds.) New York: Academic Press Parry, D.M., McMillen, I.C., Willcox, D.L.: Immunocytochemical localisation of prolactin and growth hormone in the ovine pituitary. A morphological and quantitative study. Cell Tissue Res. (in press) (1978) Parsons, J.A., Erlandsen, S.L.: Ultrastructural immunocytochemical localisation of prolactin in rat anterior pituitary by use of the unlabeled antibody enzyme method. J. Histochem. Cytochem. 22, 340-352 (1974)
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Petrali, J.P., Hinton, D.M., Moriarty, G.C., Sternberger, L.A.: The unlabeled antibody enzyme method of immunocytochemistry. Quantitative comparison of sensitivities with and without peroxidaseantiperoxidase complex. J. Histochem. Cytochem. 22, 782-801 (1974) Petrusz, P., DiMeo, P., Ordronneau, P., Weaver, C., Keefer, D.A.: Improved immunoglobulin-enzyme bridge method for light microscopic demonstration of hormone-containing cells of the rat adenohypophysis. Histochemistry 46, 9-26 (1975) Suh, H.K., Frantz, A.G.: Size heterogeneity of human prolactin in plasma and pituitary extracts. J. Clin. Endocr. Metab. 39, 328-935 (1974) Wallace, A.L.C., Ferguson, K.A.: The preparation of sheep growth hormone. J. Endocrinol. 26, 254263 (1963) Accepted December 9, 1978
