Cell Count Value of Pituitary Basophils and Serum TSH, LH and FSH Concentrations During the Short Interval after Thyroidectomy

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1 Endocrinol. Japon. 1978, 25 (1), Cell Count Value of Pituitary Basophils and Serum TSH, LH and FSH Concentrations During the Short Interval after Thyroidectomy KAZUYOSHI ARISHIMA, MASATOSHI SUZUKI, MASAO YOKOYAMA AND FUJIO YOSHIMURA Department of Anatomy, Jikei University School of Medicine, Minato-ku, Tokyo 105, Japan Synopsis Quantitative changes of pituitary basophils were investigated by the method of Chalkley, together with radioimmunoassays of serum TSH, LH and FSH concentrations, in rats during the short interval after thyroidectomy (TX)(3, 6, 12 hr, 1, 3, 5 and 7 days). The irregularly shaped or elongated II-type cells (classical thyrotrophs), which are stained intensively with thionin, immediately decrease in number below the normal value (2.7 }0.7%), to 0.6 }0.2% at 12 hr and 0.3 }0.1% at 7 days. The intermediate cells (II/III-type cells) can be easily distinguished, by their staining properties and shape, from the II-type cells and III-type cells (classical LH-gonadotrophs) during this time interval. The II/III-type cells are enlarged polygonal cells whose central area stains with PAS and whose peripheral area shows an affinity for thionin. They gradually increase in number after the first day. The total number of all kinds of the PAS-positive cells tends to rapidly increase after TX and reaches the highest value (12.3 }0.8%) after as early as 12 hr. They are distinguishable as oval III-type cells stained violet and large spherical or polygonal IV-type cells stained red. However, the values returns to normal (9.7 }0.5%) by 7 days. The total of all kinds of basophils (II-, II/III-, III-, III/IV- and IV-type cells), however, is not altered but remain balanced through the entire time. Serum TSH concentrations rise at 3 hr and fall thereafter, but rise again at 5 days (230.5 }16.7ng/ml), the latter being equivalent to twice the normal value (122.5 }8.7ng/ml). Serum FSH concentrations tend to be slightly reduced, but not profoundly, through the entire time course after TX. Serum LH concentrations quickly and conspicuously 4crease after the operation, reaching the lowest value (0.18 }0.06ng/ml), equivalent to approximately 1/4 of the normal value (1.42 }0.12ng/ml), within one day. It has been corroborated by the present results of cell counts and radioimmunoassays that, following TX, the II-type cells may transform morphokinetically into the III- and IV-type cells, supporting a working hypothesis as to the secretory cycle of the basophil proposed by Yoshimura et al.(1977). Received July 19, Abbreviation: II: II-type basophil (classical thyrotroph), irregular in shape, which stains intensively blue with thionin. III: III-type basophil (classical LH-gonadotroph), oval in shape, which stains violet combinedly with PAS and thionin. II/III: The intermediate cell between the II-and III-type basophil, whose peripheral area stains IV: blue with thionin, and whose central area stains red with PAS. It hypertrophies to be polygonal in shape. IV-type basophil (classical FSH-gonadotroph), large spherical in shape, which stains red with PAS, being characterized by the circular arrangement of Golgi apparatus. Reprint requert to Dr. F. Yoshimura.

2 ARISHIMA et al. Despite numerous light- and electronmicroscopical observations and radioimmunoassays of serum TSH concentration during the chronic interval after thyroidectomy (TX), there have been, unexpectedly, few investigations dealing with the acute response on basophils and serum TSH concentration to TX (Purves and Griesbach, 1956; Salaman, 1964; Davis and Borger, 1973). For this reason, histometrical estimations of the various types of basophils and radioimmunoassays of serum TSH, LH and FSH concentrations were carried out in this study during the short interval after TX. Soji et al.(1976) and Yoshimura et al. (1977) proposed a working hypothesis as to the secretory cycle of the basophil. According to this hypothesis, the classical TSH-, LH- and FSH-cells may not be independent but merely represent different phases in the secretory cycle of the same basophil. This hypothesis, however, lacks enough evidence of transformation of the thyrotroph into the gonadotroph. Many previous cytologists have believed in the independent nature of the two kinds of basophils. The purpose of these acute or subacute experiments of TX is to confirm whether our working hypothesis is supportable or not, in terms of the coordinated histometrical changes of three basophils and the serum concentrations of three glycoprotein hormones. Materials and Methods Wistar-Imamichi male rats of 45-day-old were surgically thyroparathyroidectomized under ether anesthesia. They were sacrificed 3, 6, 12hr, 1, 3, 5, 7 and 14days after the operation. Each group and the sham operated controls, consisted of five rats. Blood samples (about 5 cc/rat) obtained from the jugular vein under ether anesthesia, were allowed to clot at room temperature, and then centrifuged. Serum was stored at -20 Ž until assayed. The sera from the 5 rats in each group were pooled. LH and FSH concentrations in the sera were determined, using the NIAMDD-RAT-FSH-RP-1 and LH-RP-1 radioimmunoassay according to the method of Daane and Parlow (1971) slightly modified by Wakabayashi et al.(1972). TSH concentration in the sera was determined by the use of NIAMDD-RAT-TSH-RP-1. After removed, pituitary tissues were immediately embedded in paraplast, and serial horizontal sections were cut at 5-tm thickness. The secretions were stained with aldehyde thionin and counterstained with PAS (periodic acid Schiff's reaction) and orange G. The cell count was determined by the "hitting percentage" method (Chalkley, 1943) on every 20th section; the total number of sections amounting to The total number of pointed cells was calculated to be between 500 and The points of 5 needles set in the occular may hit on the four objectives: (1) thionin-positive cells,(2) PAS-positive cells,(3) thionin-pas-positive cells,(4) the other negative glandular cells. The "hitting percentage" indicates the incidence of these four cells, Results Histological findings of the various types of basophils in the normal controls Classical pituitary thyrotrophs (II) from the normal rats are stained diffusely or intensively with aldehyde thionin. They are small or lean cells, sometimes polygonal or polyhedral in shape (Fig. 1). Their shape and staining property with thionin make it possible to identify them, despite considerable variation in cell character. In some thyrotrophs (II-type cells), thionin-positive granules fill the whole cell body (Fig. 1), but in some other hypertrophic II-type cells, the Figs. 1 and 2. Central area of the pituitary from a normal male rat. ~400. Fig. 3. Peripheral area of the pituitary from a normal male rats. ~400. Figs. 4 and 5. Central area of the pituitary from a TX-rat, 12hr after the operation. ~1000. Figs. 6 and 7. Peripheral area of the pituitary from a TX-rat, 12hr after the operation. ~200, ~400. Fig. 8. Central area of the pituitary from a TX-rat, 24 hr after the operation. ~400.

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4 ARISHIMA et al. granules are accumulated at the periphery of cytoplasm representing the blue fringe and the central area is stained red with PAS (Fig. 2). The latter exhibit two staining abilities with thionin and PAS. This kind of cells may be analogous, in staining properties, with the intermediary cells (II/ III-type cells in Fig. 2) between the classical thyrotrophs and LH-gonadotrophs. The classical gonadotrophs are generally divided into two categories: LH-gonadotroph (IIItype cell) and FSH-gonadotrophs (IV-type cell). Light-microscopically, the former, oval in shape, being double stained violet with two dyes, are commonly situated at the periphery of the gland (Fig. 3). The latter are large spherical or ovoid in shape and stain red with PAS, being characterized by the definite circular arrangement of Golgi apparatus in their enlarged cytoplasm (IV in Figs. 1 and 2). The IV-type cells are scattered throughout the gland with a slight tendency to accumulate in the sex zone. Histological and histometrical findings of the various types Qf basophils during the short interval after TX The population of the II-type cells was 2.7 }0.7% in the normal control, but was rapidly reduced after TX, being equivalent to 0.6 }0.2% within 12 hr (Fig. 9). Throughout the interval of the study the population in the gland remained low (0.3 }0.1%). On the other hand, the population of the intermediate types (II/III-type cells) did not significantly increase, but showed an inclination to become elevation within one day (1.1 }0.6%). These II/III-type cells usually became hypertrophic after TX and provided with a peripheral fringe reacted with thionin (Figs. 4 and 5). The distribution of the II-type cells in the gland is demonstrated in Figure 5, which gives an impression of the low population. As seen in Figure 9, the quantitative changes of the II-type cells is in inverse relationship to those of III- and IV-type cells which reach the highest value (12.3 }0.8%) as early as 12hr, compared to the normal value (9.7 }0.5%). The accumulation of III-type cells, as demonstrated in the photomicrographs, is in the superficial area of the gland (Figs. 6 and 7), while the III- and Fig. 9. Cell count value of various types of basophils in rats during the short term after TX. Determined by the "hitting percentage" method (Chalkley, 1943) on aldehyde thionin-pas-orange G stained preparations. Vertical bars represent the mean }S.D. Fig. 10. Cell count value of various types of basophils in rats during the short term after TX. Determined by the "hitting percentage" method (Chalkley, 1943) on aldehyde thionin-pas-orange G stained preparations. Vertical bars represent the mean }S.D.

5 NUMBER OF THE BASOPHILS AFTER TX IV-type cells are found scattered quite numerously in the central area of the gland (Fig. 8). The cytological characteristics make it possible to identify both types of cells. The high population of the total classical PAS-positive gonadotrophs (III- and IV-type cells)(fig. 9) may be attributable to the progressive transformation from the II-type cells after the release of secretory granules during the short interval. The change in population of III- and IV-type cells is in mirror image with that of IIand II/III-type cells (Fig. 10). However, the total population of the basophils remains constant during the entire course of experiment (Fig. 10). Serum TSH, LH and FSH concentrations after TX In our measurement, high serum TSH concentrations were observed at 3hr and 5days. The normal value of serum TSH is }8.7ng/ml and the highest value }16.7ng/ml at 5days (Fig. 11). The serum LH concentration quickly decreases after the operation, reaching the lowest value (0.18 }0.06 ng/ml) at one day, equivalent to 1/4 of the normal value (1.42 } 0.12 ng/ml) with a trend toward recovery at 5 and 7 days. The serum FSH concentration decreases slightly, but not profound- Fig. 11. Serum TSH, LH and FSH concentrations in rats during the short term after TX. Determined by the use of NIAMDD-RAT-TSH-RP-1, NIAMDD-RAT-LH-RP-1 and NIAMDD-RAT- FSH-RP-1. Vertical bars represent the mean }S.D. ly, after the operation, but recovers after the transient decrease. Its normal value is 1020 }70 ng/ml and the lowest value 718 Discussion Regarding the change of serum TSH concentration following TX, Salaman (1964) found a triphasic response of serum TSH concentration in their long-term experiments. A peak appeared during the interval from the first to the 5th day. Davis and Borger (1973) found a linear increase in serum TSH concentration from the first to the 42nd day after TX, but no change in the mean serum LH concentration. The marked decrease in serum LH concentration at 12hr and at 1day, equivalent to 1/4 of the control value, was quite surprising. On the contrary, the PAS-positive classical gonadotrophs, III- and IV-type cells, conspicuously increased in number. This rapid and striking increase is inconsistent with the remarkable reduction in serum LH concentration. For this reason, it may be, first of all, tentatively postulated that the classical gonadotrophs may merely store a large amount of LH and FSH without being actively depleted after TX. Nevertheless, since it was electronmicroscopically observed by Kiguchi (1978) that these gonadotrophs undergo degranulation during the corresponding days after TX, the above postulation as to the storage seems to be incorrect. On the other hand, the II-type cells decreased in number in reverse of high serum TSH concentration at 12hr. Disapperance of the II-type cells may be attributable to less staining ability with thionin due to the deprivation of their secretory granules. Purves and Griesbach (1956) found a definite degranulation of thyrotrophs stained with aldehyde fuchsin (AF) within 2 days after TX. According

6 ARISHIMA et al. to them, after deprivation, the thyrotrophs lost their stainability with AF and hypertrophied to become recognizable as the preliminary TX-cells by the 6th day. According to the present histometrical study, the IIIand IV-type cells increased considerably in number in reverse relationship to a reduction in number of the II-type cells in the gland within 3 or 4 days ; the II/III-type cells slightly increased in number by 12hr or 1day. In the preceding paper, Kiguchi (1978) reported electronmicroscopic observations on the process of degranulation in the II-type cells, followed by the transformation into the III-type cells during the short interval after TX. The present histometrical results implicitly reply on the electronmicroscopic interpretation by Kiguchi that the II-type cells may transform into the III- or IV-type cells. According to the present radioimmunoassays, as shown in Figure 11, at 5 days (subacute phase), serum TSH concentrations were highest and serum LH concentrations were recovered to some extent from the preceding low value at 3 hr (acute phase); serum TSH concentrations were also high while serum LH concentrations were conspicuously lowered to the same level as revealed at 5 days (subacute phase). It is noteworthy that the ratio of the population of II- and II/III-type cells to that of III- and III/IV-type cells at 5 days corresponds to the ratio at 3hr. From the result that serum LH concentrations remained in low level at the moments of the high TSH concentration both at the acute and subacute phase after TX, a possibility may be assumed that III- and III/IV-type cells can secrete not only LH and FSH but also TSH according to a concept of secretory cycle of the basophils (Yoshimura et al., 1977). The immunohistochemical demonstration that a large part of the III- and IV-type cells which accumulated within 12hr after TX are also stained with the antibody to TSH will be reported in the subsequent paper. From the histometrical result, shown in Figure 9, that the population curves of II-type cells and II/III-type cells cross each other at the interval between 6 and 12hr, and from the histometrical result, shown in Figure 10, that the curve showing the changes of sum number of II- and II/III-type cells is in mirror image with the curve showing the sum number of III- and IV-type cells, it may be suggested that the II-type cells are not stabilized but are destined to be transformed into the III- and IV-type cells when stimulated, although many cytologists have not accepted the transformation of the thyrotroph into the gonadotroph. We must here stress again the important short-term effect of TX in lowering the serum LH concentration. This acutely lowering effect is so striking that it can be regarded as the preponderant function during the early phase of thyroxine deficiency. The only unequivocal effect of a 5-min interval ether anesthesia on LH secretion was reported by Ajika et al.(1972), Seyler and Reichlin (1973) and Reier et al. (1974) to be the transient increase within 15 min followed by the decrease within 60 min. However, Neill (1970) found no effect of ether-laporatomy on LH secretion. It was reported by Contopoulos and Koneff (1963) that pituitary FSH and ICSH concentrations increased 56 days after TX in the adult rats, and by Shima (1963) that hypothyroid status accerlated the gonadal function and hyperthyroid one suppressed it in the infant rats. Fujii (1973) reported an increase in the endogenous LH release after TX in rats sterilized with 1mg of testosterone propionate. She found the first diestrous vaginal smears developing within a few days after TX. In conclusion, TX may not only act, during the acute phase, upon the classical thyrotrophs but also upon a series of basophils. The present results confirmed a working hypothesis of Yoshimura et al.(1977) as to the secretory cycle of the basophil.

7 NUMBER OF THE BASOPHILS AFTER TX Acknowledgement The authors would express the cordical thanks to Associate Prof. K. Wakabayashi, Institute of Endocrinology, Gunma University, for his helpful advices during our performance of radioimmunoassays. Also we are grateful to Prof. M. Lee, British Columbia University in Vancouver, for his kindness in preparding this English manuscript. Radioimmunoassay reagents were kindly supplied by Dr. A. F. Parlow and the NIAMDD rat pituitary hormone program. References Ajika, K., S. P. Kalra, C. P. Fawcett, L. Krulich and S. M. McCann (1972). Endocrinology 90, 707. Chalkley, H. W.(1943). J. Nat. Cancer Inst. 4, 47. Contopoulos, A. N. and A. Ko.neff (1963). Acta Endocrin.(Kbh). 42, 275. Daane, T. and A. F. Parlow (1971). Endocrinology 88, Davis, S. L. and M. L. Borger (1973). ibid., 92, Fujii, T.(1973). Endocrinol. Japon. 20, 425. Kiguchi, Y.(1978). ibid., 25, 75. Neill, J. D.(1970). Endocrinology 82, Purves, H. D. and W. E. Griesback (1956). J. Endocrin. 13, 365. Reier, P. J., W. K. Morishige and I. Rothchild (1974). Neuroendocrinogy 16, 43. Salaman, D. F.(1964). J. Endocrin. 29, 283. Seyler, P. J. and S. Reichlin (1973). Endocrinology 81, 295. Shima, S.(1963). Jap. J. Animal Reprod. 9, 49.(in Japanese) Soji, T., S. Sato, Y. Shishiba, M. Igarashi, T. Shioda and F. Yoshimura (1977). Endocrinol. Japon. 24, 19. Wakabayashi, K., J. Antunes-Rodringnes, B. Tamaoki and M. McCann (1972). Endocrinology 90, 690. Yoshimura, F., T. Soji, T. Kumagai and M. Yokoyama (1977). Endocrinol. Japon. 24, 185.