a study of tsh-synthesis of spontaneously hypertensive rats by electron microscopic morphometry and...

Cell Tissue Res. 201,467-477 (1979) Cell and Tissue Research �9 by Springer-Verlag 1979

A Study of TSH-Synthesis of Spontaneously Hypertensive Rats by Electron Microscopic Morphometry and Autoradiography

Takashi Fujiwara

Department of Pathology, Ehime University School of Medicine, Shigenobu, Japan

Summary. In order to compare the functional state of the anterior pituitary of spontaneously hypertensive rats (SHR) with that of normotensive Wistar Kyoto rats (WKR), the anterior pituitary was examined by morphometry and autoradiography at the level of electron microscopy. The relative number and the relative volume of thyrotrophs in the anterior pituitary were significantly greater in SHR compared with age-matched WKR at 0, 7, 30-33 days, and 10 months of age, while the relative number of somatotrophs in SHR was significantly smaller at 1 and 10 months of age. Electron microscope autoradiographic analysis of uptake of 3H-lysine by thyrotrophs of both strains at the age of approximately one month showed that 3H-lysine was incorporated into protein and transported finally to secretory granules which migrated to near the cell membrane to be discharged. Silver grains were significantly more numerous over the thyrotrophs of SHR than over those of W K R at 30 min, 1 h, and 4h after the injection of 3H-lysine.

The present study has ascertained morphologically that a congenital hypersynthesis of TSH by the anterior pituitary occurs in SHR.

Key words: Anterior pituitary - Hormone synthesis - Morphometry - Autoradiography - Electron microscopy.

The spontaneously hypertensive rat (SHR) is one of the animal models for human essential hypertension (Okamoto and Aoki, 1963). An elevation of TSH levels in pituitary (Kojima et al., 1975b, 1976) and in plasma (Kojima et al., 1975a, b, 1976; Manger and Werner, 1973; Manger et al., 1974) has been reported in SHR. Enzyme

Send offprint requests to: T. Fujiwara, Department of Anatomy, Ehime University School of Medicine, Shigenobu, Ehime 791-02, Japan

Acknowledgements: The author expresses his appreciation to Professor R. Tabei and Dr. H. Mori for helpful discussion and advice in preparation of the manuscript, to Dr. Y. Uehara, Department of Anatomy, Ehime University School of Medicine, for helpful advice, and to Miss M. Terada for technical assistance

0302-766X/79/0201/0467/$02.70

468 T. Fujiwara

histochemical studies on the hypo tha lamus of SHR suggest the hyperfunct ion of m a n y hypotha lamic nuclei which were assumed to activate TSH-cells even in the prehypertensive stage (Okamoto et al., 1966). A n increase in the weight of the pi tu i tary con ta in ing more basophils in the anter ior lobe has been noted at the prehypertensive stage, with a much greater increase as hypertension persists (Aoki et al., 1963). The basophils in the anter ior pituitary, however, seem to comprise at least two distinct cell types: the thyrotrophs and gonadot rophs (Bloom and Fawcett , 1975). Therefore, an increase in the percentage ofbasophi l s in the anter ior lobe of the SHR seems no t always to coincide with the increase in the percentage of thyro t rophs and with the elevation of p lasma and pi tui tary TSH level. Electron microscopy is far more advantageous than light microscopy for morphophysiologi- cal studies of the anter ior pituitary, because it enables us to identify unambiguous ly the cell types in the pi tui tary (Kurosumi , 1968). The present study intends to compare morphological ly the ho rmone synthetic activity of the thyrot rophs of SHR with that of Wistar Kyoto rats (WKR), utilizing morphomet r ic and au toradiographic techniques at the level of electron microscopy.

Materials and Methods

Animals. Male SHR and normotensive Wistar Kyoto rats (WKR) were used in the present study. Animals were maintained on normal lab chow and tap water adlib. In the following procedures, both SHR and WKR were treated in the same way.

Electron Microscopy

1. Tissue Preparation. Animals used in the study of the anterior pituitary by morphometry were 0, 7, 15, 30-33 days and 10 months old. Each group of various ages consisted of 3 to 4 animals. After decapitation, the anterior pituitaries were fixed with a solution of 3 ~ glutaraldehyde in 0.1 M phosphate buffer, pH 7.3 for 2 h, and then postfixed with 2 ~ osmium tetroxide in identical phosphate buffer for 2 h. After block-staining with saturated uranyl acetate and dehydration in ethanol, the tissue blocks were embedded in Epon. Ultrathin sections were cut in a Porter-Blum MT-1 ultramicrotome, stained with uranyl acetate and lead citrate, and examined with a JEM 100B electron microscope. Areas of the anterior pituitary were photographed randomly at a magnification of 1,330 • and 40 photographic prints (20.3 cm • 25.4 cm) were prepared from each group of both S HR and WKR (final magnification 5,300 x ).

2. Determination of Relative Numbers of Thyrotrophs and Somatotrophs. The thyrotrophs and somatotrophs were identified and counted on prints of electron micrographs. All cells with a nuclear profile were counted. About 900-1,600 cells from each group were examined and classified into thyrotrophs, somatotrophs, and others. The percentages of thyrotrophs and somatotrophs were separately calculated. Identification of pituitary cell types was based on morphologic criteria described elsewhere (Farquhar et al., 1975; Kurosumi, 1968; Nakane, 1975).

3. Determination of Relative Volume of Thyrotrophs. The volume fraction of thyrotrophs was determined by a point counting method (Weibel and Bolender, 1973). A transparent overlay with a square lattice of lines 2 cm apart was superimposed on photographs. The number of points falling on the thyrotrophs was recorded, and its proportion to the total points was expressed as a percentage. The necessary minimal sample size was greatly exceeded in each group, when 5 ~ relative error was accepted.

TSH-Synthesis in Spontaneously Hypertensive Rats 469

Fig. 1. Electron micrograph of the anterior pituitary from 15-days-old SHR. Somatotrophs (S) and thyrotrophs (7) are observed. Thyrotrophs contain many mitochondria and well developed Golgi complex, x 4,700

Electron Microscopic Autoradiography

I. Tissue Preparation. Four rats (34-62 gm), each from SHR and WKR, about one month old, were injected intravenously through the tail with 3H-lysine (120 txCi/gm body weight) (Radiochemical Centre Amersham, England). Animals were decapitated 5 min, 30 rain, 1 h, and 4 h after administration of the label. Fixation procedures were the same as described above. Sections exhibiting light gold interference

470 T. Fujiwara

Table 1. Percentage of thyrotrophs in the anterior pituitaries of SHR and W K R at various ages

The values are Means _+ S.E.M. NS = Not significant a Significantly different from W K R (p <0.001)

Days after birth

SHR W K R

0 27.5_+ 1.18 a 20.0_+ 0.96 7 38.5_+ 1.46 ~ 29.8+ 1.56

15 35.1_+ 1.75 Ns 31.0_+ 1.52 30 27.5_+ 1.68 a 20.2_+ 1.07

300 23.3_+ 1.81 a 9.1 -+ 1.08

TaMe 2. Percentage of somatotrophs in the anterior pituitaries of SHR and W K R at various ages

The values are Means _+ S.E.M. NS = Not significant a Significantly different from W K R (p<0.05) b Significantly different from W K R (p < 0.001)

Days SHR W K R after birth

0 21.7_+ 1.97 Ns 22.6_+ 1.74 7 31.7_+ 2.10 a 39.1 _+ 2.47

15 33.1 -+ 1.98 Ns 30.7_+ 1.95 30 31.1 -+ 1.61 ~ 42.0_+ 1.86

300 38.0+ 2.18 b 60.1 + 2.60

Table 3. Percentage volume of thyrotrophs in the anterior pituitaries from SHR and W K R at various ages

The values are Means + S.E.M. NS = Not significant a Significantly different from W K R (p <0.02) b Significantly different from W K R (p < 0.001)

Days SHR W K R after birth

0 25.3+ 1.22 a 19.0_+ 1.41 7 34.7+ 1.33 b 26.7_+ 1.28

15 33.8+ 1.78 Ms 31.1 _+ 1.58 30 23.8+ 1.36 a 19.3_+ 1.29

300 12.6_+ 1.23 b 5.0+ 0.63

color (about 1,000 A thick) were mounted on collodion-coated copper grids, stained with uranyl acetate. and then coated with a thin layer of carbon. An emulsion, Sakura NR-H 2 (Konishiroku Photo Ind. Co., Japan) was applied to the sections by means of dipping. After four weeks exposure at 4~ the preparations were developed in Konidol X (Konishiroku Photo Ind. Co., Japan). Then the preparations were stained with lead citrate and covered again with a layer of carbon. Thyrotrophs were photographed from selected sections showing a distinct plasma membrane, a nucleus with clearly visible envelope, and a Golgi complex, regardless o f the number o f silver grains present on the sections. The silver grains were counted on photographic enlargements at a final magnification of 15,000 (20.3cm x 25.4cm). Background was determined by examining "cold" preparations treated in the same way as "hot" ones and was of the order of 2-3 grains per 150-mesh grid square.

2. Analysis of Autoradiographs. In order to investigate the pathway of transport of protein with incorporated 3H-lysine, the autoradiograms were analyzed according to Williams' method. This method has proven its utility in comparable studies of the hypothalamo-neurohypophysial system (Kent and Williams, 1974) and adrenal medulla (Benchimol and Cantin, 1978) which also contain small secretory granules.

Cell components were divided into a series of putative sources of radiation, termed "primary items", "junctional items", and "compound items". Each grain was alloted to 19 different items, as described


Table 4. Complete grain and circle analysis of micrographs of thyrotrophs from SHR 30min after labeling

Items No. of Actual no. Expected no. regular of silver of silver circles grains grains

if random

1. Nucleus 1,274 123 172.1 2. Nucleus/RER 184 25 24.9 3. Golgi/Cytoplasmic matrix 485 123 65.5 4. Golgi/Cytoplasmic matrix/Mitochondria 27 11 3.6 5. RER/Cytoplasmic matrix 725 97 97.9 6. RER/Cytoplasmic matrix/Mitochondria 209 26 28.2 7. Mitochondria 98 7 13.2 8. Mitochondria/Cytoplasmic matrix 119 12 16.1 9. Cytoplasmic matrix 144 13 19.5

10. Granules/Cytoplasmic matrix 349 40 47.1 11. Granules/Cytoplasmic matrix/RER 723 94 97.7 12. Granules/Cytoplasmic matrix/Mitochondria 85 11 11.5 13. Granules/Cytoplasmic matrix/Golgi 54 27 7.3 14. Granules/Cytoplasmic matrix/Cell membrane 280 49 37.8 15. Cell membrane/Cytoplasmic matrix 243 19 32.8 16. Lysosomes 1 0 0.1 17. Lysosomes/Cytoplasmic matrix 22 1 3.0 18. Multivesicular bodies 0 0 0.0 19. Multivesicular bodies/Cytoplasmic matrix 5 1 0.7

Total 5,027 679 679.0

Table 5. Observed and random silver grain distribution on grouped items of thyrotrophs of SHR and WKR at 30 min after injection of 3H-lysine

Item Grouped items SHR WKR no.

Silver Silver (observed- Silver Silver (observed- grains grains expected) 2 grains grains expected) 2

observed expected expected observed expected expected

1-2 Nucleus 148 197.0 12.19 127 162.3 7.68 3 4 Golgi complex 134 69.1 60.96 94 47.8 44.65 5-6 Rough endoplasmic

reticulum 123 126.1 0.08 103 98.2 0.23 7-8 Mitochondria 19 29.3 3.62 30 33.3 0.33 9 Cytoplasmic matrix 13 19.5 2.17 34 24.5 3.68

10-13 Granules/Cytosol 172 163.6 0.43 137 136.8 0.00 14-15 Cell surface 68 70.6 0.10 38 56.9 6.28 16-17 Lysosomes 1 3.1 1.42 0 2.4 2.40 18-19 Multivesicular 1 0.7 0.13 0 0.8 0.80

bodies

Total circles 5,027 Total circles 5,689

Total grains 679 Total grains 563

X 2 = 81.10; p<0.001 X 2 = 66.05; p<0.001

The numbers of items correspond to those enumerated in Table 4

472 T. Fujiwara

Table 6. Relative volume (RV) of secretory granules and cytosol, and relative specific radioactivity ('RSR) of secretory granules of thyrotrophs from SHR and WKR at various intervals after labeling

Time after Items SHR WKR injection (min) RV RSR RV RSR

30 Secretory 0.17 0.40 0.15 0.00 granules

Cytosol 0.83 0.85


Cytosol 0.82 0.84


Cytosol 0.84 0.85

previously (Benchimol and Cantin, 1978) (Table 4). From 486 to 687 silver grains were attributed to the organelles of thyrotrophs at each time interval.

The relative effective area of each item was measured by applying regular arrays of 4.0 mm circles to each micrograph, using a transparent overlay screen. Each circle was alloted to its appropriate item. The number of circles necessary to give a satisfactory relative effective area estimate was determined by the method of Weibel and Bolender (1973). The necessary minimal sample size was greatly exceeded for all major items, at the 5 ~ relative error level. A total of 5,027 to 5,724 circles was applied per treatment. Grain and circle frequency distributions of the different item groups were compared using the chi-square test (Table 5) in order to find out if they were different from random distributions.

3. Determination of the Relative Specific Radioactivity of Organelles. The relative specific radioactivity of cell organdies was determined by expressing each grain count and circle count as a percentage and dividing grain percentage by the corresponding circle percentage (Fig. 4).

4. Determination of the Relative Specific Radioactivity of Secretory Granules. Subanalysis of the compound item granule/cytosol (Item no. 10-13 in Table 4) was performed by combining silver grain and effective area data with volume fractions derived by a point-counting method (Weibel and Bolender, 1973), utilizing a regular point pattern on a transparent overlay (Table 6). A total of 4,769 to 5,473 points was applied per treatment. The relative specific radioactivity (RSR) of the secretory granules was calculated as shown below. RSR of secretory granules =

RSR of granule/cytosol - [Relative volume of cytosol x RSR of cytosol]

Relative volume of secretory granules

where the cytosol comprises rough endoplasmic reticulum, Golgi complex, mitochondria, free ribosomes, and cytoplasmic matrix.

5. Comparison of Silver Grain Counts over Thyrotrophs. Silver grains over the cytoplasm of each 50 thyrotrophs from SHR and WKR were counted on electron micrographs (Table 7).

Results

T h e t h y r o t r o p h s in t h e a n t e r i o r p i t u i t a r i e s o f 1 - m o n t h - o l d a n d I 0 - m o n t h s - o l d S H R

a n d W K R s h o w e d s o m e c h a r a c t e r i s t i c f ea tu re s . T h e y a re cells o f m o d e r a t e size,

s o m e w h a t a n g u l a r in s h a p e , w i t h o c c a s i o n a l p r o c e s s e s p r o t r u d i n g b e t w e e n

n e i g h b o r i n g cells. T h e c y t o p l a s m c o n t a i n s s m a l l o v o i d o r r o d - s h a p e d m i t o c h o n -


Table7. Silver grain counts over the cytoplasm of thyrotrophs from SHR and WKR at various intervals after injection of aH- lysine

Incorporation SHR WKR time (min)

5 5.6___ 0.34 Ns 4.8"4- 0.37 30 9.7"4- 0.81 a 7.6"4- 0.64 60 9.0"4- 0.59 b 6.0"4- 0.55

240 8.1-4- 0.52 a 6.6"4- 0.51

The values are Means + S.E.M. of the number of silver grains per cell NS = Not significant a Significantly different from WKR (p < 0.05) b Significantly different from WKR (p <0.001)

dr ia , sca t tered r i b o s o m e s wi th some p o l y s o m a l clusters , a well deve loped Go lg i complex , some prof i les o f r o u g h e n d o p l a s m i c r e t i c u l u m (RER) , a n d p o p u l a t i o n o f m o d e r a t e l y dense , r o u n d g ranu l e s 100-180 n m in d iamete r . T h e s o m a t o t r o p h s , which were m o s t a b u n d a n t cells in the an te r io r p i tu i tary , were oval or p o l y g o n a l in shape a n d c o n t a i n e d a Go lg i complex , a b u n d a n t R E R a r r a n g e d in l ame l l a t ed

Fig. 2. Electron microscopic autoradiogram of a thyrotroph (TSH) in the anterior pituitary from SHR 1 h after the injection of 3H-lysine. Silver grains are observed over Golgi complex (Go) and secretory granules (SG), and near the cell membrane (arrow). x 10,000

474 T. Fujiwara

Fig. 3. Electron microscopic autoradiogram ofa thyrotroph ofWK_R 4 h after the injection of aH-lysine. Silver grains are observed over secretory granules (SG) and near the cell membrane (arrow). x 10,000

stacks, some mitochondria and a large number of electron dense secretory granules. The secretory granules of the somatotrophs were round and 300M00nm in diameter. Compared to these adult rats, the thyrotrophs and somatotrophs of newborn rats had more free ribosomes, a smaller Golgi complex, a less developed RER, and a smaller number of secretory granules. But two cell types were clearly identified at any postnatal age by their ultrastructural features (Fig. 1).

A comparison of the percentages of thyrotrophs and somatotrophs of SHR and WKR at different ages is shown in Tables 1 and 2. The percentage of thyrotrophs in SHR was greater than in WKR at all ages examined, whereas that of somatotrophs was smaller in SHR than in WKR at 1 month and I0 months of age (p < 0.001).

Table 3 shows a comparison of the percentage volume occupied by thyrotrophs in the anterior pituitary from SHR and WKR at various ages. The relative volume of thyrotrophs in the anterior pituitary was also greater in SHR than in WKR (p<0.02), although at 15 days of age the value for SHR was not significantly different from that for WKR.

In electron microscopic autoradiographs a few silver grains were observed over the nucleus and the cytoplasm of thyrotrophs in both SHR and WKR as rapidly as 5 min after the injection of 3H-lysine. The cytoplasmic grains were related primarily to RER and Golgi complex. The distribution of silver grains over various organelles was analyzed in thyrotrophs at 30 min, 1 h, and 4 h after the injection (Figs. 2, 3). At all times examined, the grain distribution differed from a random one (p < 0.001) as


Rough end oplasmic Goigi Secretory

ret iculum complex granules Cell surface

i t )

1t lla BIll s g

_1 30 60 240 30 60 240 30 60 240 30 60 240

Time after injection (min)

Fig. 4. Average relative specific radioactivity of cell organdies of thyrotrophs from SHR and WKR at various intervals after injection of 3H-lysine. The open bars represent WKR and the hatched bars SHR

determined by the chi-square test (Table 5). Figure 4 shows an average relative specific radioactivity of organelles, which varied noticeably with time of incorporation of aH-lysine. The relative specific radioactivity decreased steadily in RER. The radioactivity decreased markedly in Golgi complex up to 4 h after the injection, while it increased very rapidly in secretory granules at 1 h to reach a maximum at 4 h. At the cell surface, the radioactivity increased rapidly at 4 h. Secretory granules had the highest radioactivity of all the organelles at 4 h after the injection.

Table 7 shows a comparison of the number of silver grains over the thyrotrophs of SHR and WKR at various times after the injection of aH-lysine. The number of silver grains over the cytoplasm of thyrotrophs was greater in SHR than WKR at 30min, 1 h, and 4h after the injection of label (p<0.05).

Discussion

There have been very few observations of the relative number or the relative volume of anterior pituitary cells, partly because of difficulty in identifying the cell types with certainty at the level of light microscopy. Recently, however, Surks and DeFesi (1977) reported on the relative numbers of anterior pituitary cells in euthyroid and hypothyroid rats at the level of electron microscopy. Stratmann et al. (1972) also described a change in the distribution of cell types in the normal and propylthiouracil-treated anterior pituitary. These groups found that thyrotrophs and somatotrophs represent 10.7 ~ and 55.3 ~ (Surks and DeFesi, 1977), or 9.1 and 66.8 ~o (Stratmann et al., 1972) of the total cell population in normal rats. The percentages of thyrotrophs and somatotrophs of WKR at 10 months of age, estimated in the present study, are 9.1 and 60.1, respectively. This result is thus in good agreement with these previous reports.

476 T. Fujiwara

The relative number and the relative volume of thyrotrophs in the anterior pituitary were both found in the present morphometric study to be larger in SHR than in WKR. On the contrary, the relative number of somatotrophs is smaller in SHR at more than one month of age, compared with age-matched WKR. These differences are exaggerated in adult animals. In the anterior pituitary of 10-months- old SHR thyrotrophs are approximately 2.5 times more numerous and larger in volume than those of WKR, whereas the relative number of somatotrophs in SHR is smaller than WKR by the factor of 0.63. Since the size of an individual thyrotroph in SHR seems to be not much different from that in age-matched WKR, and some increase in the weight of the pituitary has been reported in SHR compared with WKR (Aoki et al., 1963), it seems likely that the number, as well as the volume, of thyrotrophs per animal is larger in SHR than in WKR throughout life.

Electron microscopic autoradiography has been widely used to investigate the intracellular pathway, the time course and the localization of labeled substance in various organs. The pathway and kinetic timetable of intracellular transport of the protein moiety of the secretory product of thyrotrophs found in this study are similar to those of thyrotrophs (Pelletier and Puviani, 1973), somatotrophs (Hiura et al., 1976; Hopkins and Farquhar, 1973; Howell and Whitfield, 1973), gonadotrophs (Pelletier, 1974), and mammotrophs (Farquhar et al., 1978) reported previously. The present results apparently support the generally accepted idea in which labeled precursor is incorporated into proteins in RER, and transported through Golgi complex to secretory granules which then migrated to near the cell membrane to be released. The number of silver grains in autoradiograms may indicate the rate of hormone synthesis by thyrotrophs, since the autoradiographic analysis of intracellular transport suggests that 3H-lysine is incorporated into protein, and transported to secretory granules which contain thyroid stimulating hormone (Fig. 4). The number of silver grains observed over the cytoplasm of the thyrotrophs is greater in SHR than in WKR (Table 7). Therefore, the activity of TSH production by the thyrotrophs in SHR seems to be higher than in WKR. The relative specific radioactivity of both secretory granules and the cell surface of SHR is as high as that of WKR at 4h after the injection, while it is higher in SHR at 30min and 60 min after the injection (Fig. 4). Therefore, it is probable that the labeled material in the secretory granules is released more rapidly from the thyrotrophs of SHR than from those of WKR.

These results indicate that the anterior pituitary of SHR is in a state of hypersynthesis and probably of hypersecretion of TSH, and that this abnormality of the anterior pituitary of SHR may occur at a prehypertensive stage. The present study ascertained morphologically that the TSH level in SHR pituitary is elevated (Kojima et al., 1975b, 1976), and this may result in the elevation of plasma TSH (Kojima et al., 1975a, b, 1976; Manger and Werner, 1973; Manger et al., 1974).

References

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Bloom, W., Fawcett, D.W.: Hypophysis. In: A textbook of histology. (W. Bloom and D.W. Fawcett, eds.), pp. 503-524. Philadelphia-London-Toronto: W.B. Saunders Co. 1975

Farquhar, M.G., Skutelsky, E.H., Hopkins, C.R.: Structure and function of the anterior pituitary and dispersed pituitary cells. In vitro studies. In: Ultrastructure in Biological Systems. Vol. 7 (A.J. Dalton and F. Haguenau, eds.), pp. 84-136. New York: Academic Press 1975

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)

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Hopkins, C.R., Farquhar, M.G.: Hormone secretion by cells dissociated from rat anterior pituitaries. J. Cell Biol. 59, 276-303 (1973)

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Kent, C., Williams, M.A.: The nature of hypothalarno-neurohypophyseal neurosecretion in the rat. A study by light- and electron microscope autoradiography. J. Cell Biol. 60, 554-570 (1974)

Kojima, A., Kubota, T., Sato, A., Yamada, T., Yamori, Y., Okamoto, K.: Congential abnormality of pituitary-thyroid axis in spontaneously hypertensive rats (SHR) and stroke-prone rats (SPR). Proc. Soc. Exp. Biol. Med. 150, 571-573 (1975a)

Kojima, A., Takahashi, Y., Ohno, S., Sato, A., Yamada, T., Kubota, T., Yamori, Y., Okamoto, K.: An elevation of plasma TSH concentration in spontaneously hypertensive rats (SHR). Proc. Soc. Exp. Biol. Med. 149, 661-663 (1975b)

Kojima, A., Kubota, T., Sato, A., Yamada, T., Harada, A., Utsumi, M., Sakoda, M., Baba, S., Yamori, Y., Okamoto, K.: Abnormal thyroid function in spontaneously hypertensive rats. Endocrinology 98, 1109-1115 (1976)

Kurosumi, K.: Functional classification of cell types of the anterior pituitary gland accomplished by electron microscopy. Arch. Histol. Jpn. 29, 329-362 (1968)

Manger, W.M., Werner, S.C.: Presumably hypothyroidism in the spontaneously hypertensive rat. Fed. Proc. 32, 749 (1973)

Manger, W.M., Werner, S.C., Freedman, L.S., Dufton, S., von Estorff, I.: Multiple differences between spontaneously hypertensive (SHR) and normotensive male Kyoto Wistar rats (K.). Fed. Proc. 33, 543 (1974)

Nakane, P.K.: Identification of anterior pituitary ceils by immunoelectron microscopy. In: Ultrastructure in Biological Systems. Vol. 7 (A.J. Dalton and F. Haguenau, eds.), pp. 45-63. New York: Academic Press 1975

Okamoto, K., Aoki, K.: Development of a strain of spontaneously hypertensive rats. Jpn. Circ. J. 27, 282-293 (1963)

Okamoto, K., Tabei, R., Nosaka, S., Fukushima, M., Yamori, Y., Matsumoto, M., Yamabe, H., Morisawa, T., Suzuki, Y., Tamegai, M.: Enzyme histochemical studies on the hypothalamus of spontaneously hypertensive rats, with special reference to that of rats subjected to various endocrine interferences. Jpn. Circ. J. 30, 1483-1506 (1966)

Pelletier, G.: Autoradiographic studies of synthesis and intracellular migration of glycoproteins in the rat anterior pituitary gland. J. Cell Biol. 62, 185-197 (1974)

Pelletier, G., Puviani, R.: Detection of glycoproteins and autoradiographic localization of 3H-fucose in the thyroidectomy cells of rat anterior pituitary gland. J. Cell Biol. 56, 600-605 (1973)

Stratmann, I.E., Ezrin, C., Wellers, E.A., Simon, G.T.: The origin of thyroidectomy cells as revealed by high resolution radioautography. Endocrinology 90, 728-734 (1972)

Surks, M.I., DeFesi, C.R.: Determination of the cell number of each cell type in the anterior pituitary of euthyroid and hypothyroid rats. Endocrinology 101, 946-958 (1977)

Weibel, E.R., Bolender, R.P.: Stereological techniques for electron microscopic morphometry. In: Principles and techniques of electron microscopy. Biological applications. Vol. 3 (M.A. Hayat, ed.). pp. 237-296. New York-Cincinnati-Toronto-London-Melbourne: Van Nostrand Reinhold Co. 1973

Accepted July 14, 1979

a study of tsh-synthesis of spontaneously hypertensive rats by electron microscopic morphometry and...

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