Quantitative development of the subcommissural organ in hypothyroid mice

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Brain Research, 331 (1985) 348- 352 Elsevier

BRE 20701

Quantitative development of the subcommissural organ in hypothyroid mice ROMUALDO FERRES-TORRES, AGUSTIN CASTAIqEYRA-PERDOMO and JOSE RAMOS-NAVARRO Departamento de Anatomia, Facultad de Medicina, Universidad de La Laguna, La Laguna, Tenerife (Spain)

(Accepted November 6th, 1984) Key words: subcommissural organ - - ependyma - - hypothyroidism - - development - - karyometry - - mouse

The postnatal development of the global volume of the subcommissural organ (SCO) and of the karyometric changes of the ependymocytes in the SCO and the adjacent ventricle is studied in male albino mice aged from 25 to 160 days, and in a hypothyroid group treated with propylthiouracil with and without interruption of the treatment at 35 days. Hypothyroidism produces a decrease of the global volume of the SCO and of the nuclear size of the ependymocytes in the SCO and the adiacent ventricle.

The subcommissural organ (SCO), a secretory circumventricular organ, participates through Reissner's fiber in the regulation of the composition of the CSF in the central canal, providing catecholaminergic substances to the latter and/or removing them 6. The relationship between the S C O and the endocrine system has r e p e a t e d l y been considered (see LeonhardS and Sterba 10, for reference). Talanti 14 demonstrated in adult male rats treated with thiouracil a significant decrease of the nuclear cross section areas of the ependymal cells of the SCO and the adjacent thalamic ventricle, and described a tendency to return to normal values after interrupting the treatment. In a study of the spontaneous d e v e l o p m e n t of the SCO and the effects of castration 3-5, we described significant regressive changes in the global volume of the SCO as well as in the individual nuclei of its ependymal cells, expressed in a decrease of the nuclear m e m b r a n e (nuclear perimeter) which was absent in the adjacent ependyma. So far, there are no quantitative data about the effects of hypothyroidism on the postnatal d e v e l o p m e n t of the SCO. In the present paper we describe by karyometric procedures the global reaction of the SCO, the reaction of its ependymocytes and that of the thalamic e p e n d y m a to hypothyroidism p r o d u c e d by the administration of pro-

pylthiouracil during postnatal development. Eighty-seven male albino mice were divided into one control group and two experimental groups. The control group consisted of 42 animals aged 25, 35, 45, 55, 85, 100 and 160 days, each age group comprising 6 animals. These mice were the same as used in previous studies3,4. In the first experimental group, 25 mice were treated with propylthiouracil, which was added to the standard pellet diet at a concentration of 0.3%, and to the drinking water at a concentration of 0.002%. The t r e a t m e n t began immediately after birth, by intake by the lactating mother, and was terminated at the age of 35 days of life. The animals were sacrificed at 45, 55, 85, 100 and 160 days from birth, with 5 animals within each age-group. The second experimental group comprised 20 mice the treatment of which was not interrupted, and which were sacrificed at the ages of 25, 35, 55 and 100 days of life. After fixation by perfusion with Bouin's fluid, the brain, thyroid gland, testicles and other organs were removed and postfixed for 24 h and e m b e d d e d in paraffin under standard conditions. The brains were cut at 10 ~tm in a coronal plane and stained by the Kliav e r - B a r r e r a method; the other organs were stained with hematoxylin-eosin. The activity of the mice was d e t e r m i n e d with the aid of a gyroscope ( A p e l a b , J.C.

Correspondence: R. Ferres-Torres, Departamento de Anatomia, Facultad de Medicina, Universidad de La Laguna, La Laguna, Tenerife, Spain.

0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

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Fig. 1. Development of the global volume of the SCO in normal and thiouracil-treated mice. Montagne), by counting the number of revolutions per 15 rain. All animals were weighed before sacrifice. The nuclei of 60 ependymocytes, both of the SCO and the adjacent thalamic ventricle, were measured with the aid of a Leitz Image Analysis System (ASM), at a magnification of 2675 in the projection on the tracing board. The nuclear parameters analyzed were: perimeter, area, maximum diameters and the form factor (perimeter/area ratio), an index given by the ASM which expresses the approximation of the nuclear shape to a circle, with the maximum value being 1 and corresponding to a circle, and any deviations of the latter being expressed in values lower than 1. We have also determined the global volume of the SCO. To arrive at this measure the surface of the SCO was determined at one of each 6 sections of 10 ~m and was multiplied by the height of the 6 sections, that is b37 60. We then proceeded to add together all these partial volumes and the global volume of the organ was reached. In addition, the nuclear values of thyroid follicular cells were measured, and the seminiferous tubules were qualitatively and quantitatively studied. For statistical evaluation, the t-test and a simple analysis of variance were used.

Development of weight, activity and thyroid follicles. The weight of the control animals progressively increased up to the 50th day, thereafter remaining constant. In the experimental groups, the weight in-

creased up to the 55th day, stabilizing in the group in which treatment was not interrupted, and reaching control values in the other group, after interruption of the treatment. Compared to the control group, the hypothyroid groups exhibited significantly lower values until the 55th day. The activity was lower in both experimental groups at all ages, the differences with the control group becoming more pronounced with increasing age. The karyometric development of the thyroid follicular cells and the global volume of the thyroid gland underwent in all experimental animals the known, statistically highly significant increase, which was more pronounced in those animals in which the treatment was not interrupted. The latter presented, during the last days of life, serious respiratory difficulties caused by the goiter and the consequent tracheal compression. SCO. In those experimental animals in which the treatment was interrupted at the age of 35 days, the global volume of the SCO exhibited a significantly different development in comparison to the control group. The initial values were significantly (Fig. 1) lower, with P < 0.01, at the 25th day, and in a nonsignificant manner at the 35th and the 45th day. From the age of 55 days, there was no difference between this group and the control one. No difference was found between the group in which treatment was interrupted and the other one, in which it was continued until the day of sacrifice. In the hypothyroid ani-

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reals the perimeter and the area of the nuclei of the SCO-ependymocytes (Fig. 2) were lower at all ages studied, the difference being significant at the ages of

35 and 55 days, and nearly significant (P < 0.1) in the other age-groups. The form factor showed higher values in the experimental groups, though in a statis-

351 tical significant manner only at 85 and 160 days. No significant differences were found between the nuclear perimeters, areas and form factors of both experimental groups. Adjacent ependyma. The nuclear development of the adjacent ependyma was similar to that of the SCO: the values were lower in the hypothyroid groups than in the control group, though in a significant manner only in the first age groups, 25, 35, 45 and 55 days. Thereafter the values were nearly identical to the control ones, with the exception of the nuclear area at the age of 160 days, which was highest in the experimental group with interrupted treatment. Gonadal development. The qualitative and quantitative study of the seminiferous tubules in the control group showed that puberty occurred around the 35th day. In the hypothyroid animals, gonadal maturation took place around the 55th day of life. The study of the thyroid follicular epithelium in both experimental groups demonstrated the characteristic hypertrophy of the thyroid gland, expressed by statistically highly significant values at all ages, most evident in the group with continued treatment. The motor activity was significantly lower in all experimental age groups, another feature of hypothyroidism. In a previous paper 3 we reported an increase of the nuclear membrane and of the nuclear area of the SCO-ependymocytes, as well as an increase of the global volume of the SCO, especially in the days after puberty. The gonadectomy demonstrated the necessity of the presence of androgenic hormones in the development of the SCO, a fact that could permit consideration of this structure as a target center of these hormones 4. In the same study we did not detect any karyometric changes in the nuclei of the ependymocytes in the adjacent thalamic ventricle after androgenic deprivation. Thompson15, by the method of immunoreactive peroxidase, localized prolactin in the adjacent ependyma, but not within the SCO. Stumpf and Sarlm2, by autoradiographic methods, observed triiodothyronine in the organum vasculosum laminae terminalis, the choroid plexus, ependymocytes of discrete regions of the telencephalic and diencephalic ventricles, and in the infundibular and optic recesses, but not in the SCO. Our results show

that chemical hypothyroidism produces a decrease of activity in the ependyma of the SCO and the adjacent ventricle, in a global and apparently unspecific manner. This type of hypofunction contrasts with the effects of gonadal deprivation in male mice 4, which were localized in the SCO but not in the adjacent ependyma. The effects of castration were more evident at the days around and after puberty (35, 45, 55 days) and much more pronounced than the effects of chemical hypothyroidism, a fact that indicates a less specific character of the latter. However, it should be noted that the hypothyroid animals presented a considerable delay of puberty, with the consequent possibility that the effects of hypothyroidism were potentiated by the retardation of gonadal maturation. Our results confirm in the mouse those of Talanti 14 in male adult rats treated with methyl-thiouracil. This author described a significant decrease of the nuclear cross section area of the ependymocytes in the SCO and the adjacent ependyma, and a recovery of normal values after the interruption of the treatment with thiouracil. Our results in the experimental group in which the treatment was interrupted gave no evidence of a recovery in the SCO or in the adjacent ependyma, a fact that shows the irreversible character of the postnatal and infant hypothyroidism. The direct serotoninergic innervation of the SCO-ependymocytes and the presence of synapses type Gray I has been demonstrated1,2,9,13,16, as well as the inhibitory character of this innervation. Aside from terminals with a morphologically serotonergic nature, Bouchaud 1 described other terminals, containing an unknown transmitter substance, possibly with a cholinergic, excitatory function. There is no evidence of a direct relation between the SCO and the catecholaminergic fibers which traverse the myelinated fibers of the posterior commissure~. In addition, intraperitoneal administration of T R H increases the activity of the reticular formation of the brainstem 7. From our results and the precited bibliographic data we may conclude that the ependyma of the SCO, by a probably nervous, basically serotoninergic mechanism, responds to hypothyroidism with a morphofunctional inhibition, which is attenuated after the delayed onset of puberty.

352 1 Bouchaud, C., Evidence for a multiple innervation of subcommissural ependymocytes in the rat, Neurosci. Len., 12 (1979) 253-258. 2 Bouchaud, C. and Arluison, M., Serotoninergic innervation of ependymal cells in the rat subcommissural organ. A fluorescence electron microscopic and autoradiographic study, Biol. Cell., 30 (1977) 61-64. 3 Castafieyra-Perdomo, A., Ferres-Torres, R. and Meyer, G., Karyometric changes in the subcommissural organ of male mice after gonadectomy, Neurosci. Lett., 39 (1983) 27-31. 4 Castafieyra-Perdomo, A., Ferres-Torres, R. and Meyer, G., Cariometria del 6rgano subcomisural en desarrollo (Un estudio en el rat6n albino), Morfol. Norm. Patol., 7 (1983) 1-10. 5 Castafieyra-Perdomo, A., Meyer, G. and Ferres-Torres, R., Development of the subcommissural organ in the albino mouse (a Golgi study), J. Hirnforsch., 24 (1983) 363-370. 6 Diederen, J. H. B., Vullings, H. G. B., Rombout, J. H. W. M. and de Gunst-Schoonderwoerd, A. T., The subcommissural organ-liquor fibre complex: the binding of catecholamines to the liquor fibre in frogs of the rana esculenta complex, Acta ZooL, 64 (1983) 47-53. 7 Kor~inyi, L., Whitmoyer, D. I. and Sawyer, C. H., Effect of thyrotropin-releasing hormone, luteinizing hormone-releasing hormone and somatostatin on neuronal activity of brain stem reticular formation and hippocampus in the female rat, Exp. Neurol., 57 (1977) 807-816. 8 Leonhard, H., Ependym and circumventrikul~ire organe. In A. Oksche (Ed.), Neuroglia L Handbuch der mikrosko-

pischen Anatomie des Menschen, 4. Band, Nervensystem, 10. Teil, Springer Verlag, Berlin, 1980, pp. 177-666.

9 M01tghrd, K. and Wicklund, L., Serotoninergic synapses on ependymal and hypendymal cells of the rat subeommissural organ, J. Neurocytol., 8 (1979) 445-467. 10 Sterba, G., Das Subkommissuralorgan. In G. Sterba and W. Bargmann (Eds.), Cirkumventrikulare Organe, Leopol-

dina Symp., Schloss Reinhardsbrunn, Nova Acta Leopoldina, Suppl. 9, Deutsche Akademie der Naturforscher, Halle, 1977, pp. 103-114. 11 Stumpf, W. E. and Sar, M., Localization of thyroid hormone in the mature rat brain and pituitary. In W. E. Stumpf and L. D. Grant (Eds.), Anatomical Neuroendocrinology, Karger, Basel, 1975, pp. 318-327. 12 Stumpf, W. E. and Sar, M., Anatomical distribution of estrogen, androgen, progestin, corticosteroid and thyroid hormone target sites in the brain of mammals: phylogeny and ontogeny, Amer. Zool., 18 (1978) 435-445. 13 Takeuchi, Y. and Sano, Y., Serotonin distribution in the circumventricular organs of the rat. An immunohistochemical study, Anat. Embryol., 167 (1983) 311-319. 14 Talanti, S., Effects of thiouracil, excess thyroxine and thyroidectomy on the ependymal cells with special reference to the subcommissural organ of the rat, Anat. Rec., 159 (1967) 379-386. 15 Thompson, A., Localization of immunoreactive prolactin in ependyma and circumventricular organs of rat brain, Cell Tiss. Res., 225 (1982) 79-93. 16 Wicklund, L. and M¢llghrd, K., Neurotoxic destruction of the serotoninergic innervation of the rat subcommissural organ is followed by reinnervation through collateral sprouting of non-monoaminergic neurons, J. Neurocytol., 8 (1979) 469-480.