by an increase in the ovarian content of acid mucopolysaccharides with a sub sequent tendency to develop polycystic ovaries (Thorsee 1961).

Transcription

1 Connective Tissue Laboratory (Prof. G. Asboe-Hansen, M. D.), University Institute of Medical Anatomy, Copenhagen INHIBITION OF OVULATION AND CHANGES IN OVARIAN MUCOPOLYSACCHARIDES INDUCED BY THYROIDECTOMY IN RABBITS By Hans Thors\l=o/\e ABSTRACT Radical surgical thyroidectomy in rabbits, restricts the ability of the ovaries to ovulate following mating. Nine out of 20 thyroidectomized rabbits had severe polycystic ovaries. The ovaries of mated thyroid\x=req-\ ectomized rabbits had a higher content of water, corresponding to a given hexosamine concentration, than the ovaries of non-mated thyroidectomized, and of intact rabbits. After ovulation, the follicular fluid changed from metachromatic to ohochromatic in the thyroidectomized rabbits. These findings are discussed in relation to the theory that pre-ovulatory growth of the follicles is due to an increase in the colloid-osmotic pressure within the follicles caused by enzymatic depolymerization of acid mucopolysaccharides. It has been repoed previously that radical surgical thyroidectomy is followed by an increase in the ovarian content of acid mucopolysaccharides with a sub sequent tendency to develop polycystic ovaries (Thorsee 1961). In 1958 Zachariae 8c Jensen advanced the theory that the increase in colloid-osmotic pressure resulting from enzymatic degradation of acid muco polysaccharides in the follicular fluid played an impoant role in the ovu latory mechanism. The present experiments were designed to investigate whether changes in ovarian acid mucopolysaccharides following thyroidectomy were accompanied by changes in ovulationcapacity.

2 MATERIAL AND METHODS The experiment, carried out in late spring, comprised 45 female albino rabbits of exactly the same age and of an average weight of approximately 2000 g. The rabbits were separated sholy after bih and fed a uniform, adequate laboratory diet. At the age of 18 weeks, 25 were thyroidectomized, while 20, which were to serve as controls, had a sham operation on the anterior cervical region. Ten to foueen days later, the completeness of thyroidectomy was verified by ''"I uptake studies according to the technique previously described (Thorsoe 1961). Five showed incomplete thyroid ectomy. One rabbit died after the sham operation. Thiy days after the operations, ten thyroidectomized and nine sham-operated rabbits were mated, while ten thyroid ectomized and ten sham-operated rabbits served as controls. Mating has been found to induce ovulation in about ten hours (Walton Se Hammond 1929). Ten hours after the mating, laparotomy was carried out under Nembutal anaesthesia. The ovaries were examined for burst follicles and removed. Ovarian wet weight was determined within five minutes. Each ovary was divided into two pas, one for histological examination and one for biochemical analysis. Histological studies: One half of each ovary was fixed in a 4 /o aqueous solution of lead subacetate, embedded in paraffin, sectioned and stained with a J /o aqueous solution of toluidine blue (py 4.5-5) for half an hour. With this method the acid tissue mucopolysaccharides are visualized by metachromasia. Biochemical analysis was performed on one-half of each ovary. The water content of the ovaries was determined as the difference in weight after drying in a vacuum desiccator of the Edwards type for 48 hours under a pressure of 4-8 mm Hg at room temperature. In order to gain an impression of the acid mucopolysaccharide content, the hexos amine concentration was determined by the Elson Sc Morgan (1933) method in the modification of Boas (1953) and Kirk Sc Dyrbye (1956). The values are given as µg hexosamine per mg dried ovarian tissue. RESULTS Five out of ten thyroidectomized and eight out of nine sham-operated rabbits ovulated following mating. The five thyroidectomized rabbits that failed to ovulate were found to have polycystic ovaries. Microscopic examination of the ovaries revealed that the follicular fluids from the mated rabbits which had ovulated had changed from metachromatic to ohochromatic. In the non-mated sham-operated rabbits, as well as in the one sham-operated rabbit that failed to ovulate after mating, the follicular fluids were highly metachromatic. The ovaries of the non-mated thyroid ectomized rabbits contained numerous large metachromatic follicles. In four rabbits of this latter group, the ovaries consisted chiefly of metachromatic cystic follicles, but the intensity of metachromasia was fainter than in normal Graafian follicles. The ovaries of the mated thyroidectomized rabbits which failed to ovulate consisted mainly of cystic follicles with a faint metachromasia. These rabbits also showed apparently normal follicles, but the metachromasia had not changed to ohochromasia.

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4 Table 2. Concentration of water in non-mated thyroidectomized and sham-operated as compared with mated thyroidectomized and sham-operated rabbits. Non-mated Mated Increase, per cent P-value Controls 78.1 ± 0.9* 81.1 ±0.5* > > 0.01 Thyroidectomized 82.4 ± ± > > 0.02 Mean ± standard deviation of mean Hexosamine ug/mg dried tissue Fig. 1. Relation between water and hexosamine in ovaries from non-mated and mated thyroidectomized as compared with non-mated and mated sham-operated rabbits. Regression coefficients = b. Non-mated controls b=2.38 Mated controls = 3.o3 0 Non-mated thyroidectomized b = 3.21 Mated thyroidectomized b * 2.33

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6 Table 1 shows that the ovarian weight following mating is higher in thyroid ectomized than in intact rabbits, due primarily to increased water content. The increased water content was accompanied by an increase in the content of total hexosamine. Ten hours after mating, the water content was significantly increased in the ovaries of normal as well as of thyroidectomized rabbits (Table 2). The relation between water and hexosamine contents in the individual rabbits is shown in Fig. 1 and Table 3. This table shows which rabbits had polycystic ovaries and failed to ovulate. Statistical analysis of the four lines on Fig. 1, by the method of least squares revealed that all four lines were parallel, the t-value for all of them being below 2. Testing of the difference between the two y-values corresponding to a given x-value, showed no signi ficant difference between the y-value on the regression line for normal rabbits and the y-value for normal mated rabbits at point 3.5. The difference between the two y-values on the lines representing normal and thyroid ectomized rabbits at point 3.8 was also not significant. On the other hand, there was a significant difference between the points on the two parallel lines representing normal and mated thyroidectomized rabbits at 4.1. There was also a significant difference between the two y-values on the lines representing thyroidectomized and thyroidectomized mated rabbits at 4.6. = In intact as well as thyroidectomized animals, an increase in the hexosamine concentration is accompanied by an increase of water in the ovaries. Evidently, after mating changes take place in the ovaries involving an increase of water in relation to a given hexosamine concentration. In the intact animals there was only a tendency towards an increase, but in the thyroidectomized animals a significant increase in the content of water per hexosamine weight-unit was demonstrated. DISCUSSION In 1944 it was demonstrated by Chu that hypothyroid rabbits showing the follicular hyperophy described by Hofmeister (1894) did not ovulate. Frederikson 8c Rydin (1947) found that only 6 out of 19 hypothyroid rabbits ovulated following mating. However, these findings were not confirmed by Krohn (1950), who found no increase in the number or size of the follicles after thyroidectomy. According to the present experiments, ovulation in hypothyroid rabbits appears to be dependent on the degree of follicular hyperophy. Rabbits with polycystic ovaries do not ovulate after mating, even though their ovaries contain, in addition to the cysts, apparently normal Graafian follicles. As demonstrated by Wislocki et al. (1947); Braden (1952), and Zachariae Sc Jensen (1958), the follicular fluid changes from metachromatic to ohochroma-

7 tic immediately before ovulation. Zachariae Se Jensen (1958) also found that this could not be due to dilution or inhibited production of mucopolysaccharides. Jensen Sc Zachariae (1958) isolated an enzyme with all the propeies of testicular hyaluronidase from follicles which were about to ovulate. They als» found an increase in colloid-osmotic pressure up to three-fold values prior to ovulation and concluded that the pre-ovulatory growth of the follicle was caused by influx of water due to enzymatic depolymerization of the acid mucopolysaccharides, a process which is fuher facilitated by increased per meability of the blood-follicular fluid barrier (Zachariae 1958). A three-fold increase in colloid-osmotic pressure has been questioned to take place merely because of depolymerization of the acid mucopolysaccharides which are present in the follicular fluid in a concentration of only /o (Marcker, pers. comm. 1961). The present findings do not permit the conclusion that the increased con centration of hexosamine and the presumed higher concentration of acid muco polysaccharides are essential factors in the increased binding of water after mating. Taking into account, however, the simultaneous alteration of the follicular fluid from metachromatic to ohochromatic, it seems likely that the mucopolysaccharides of the follicular fluid undergo depolymerization prior to ovulation. ACKNOWLEDGEMENTS These investigations were aided by grants from the Population Council, Rockefeller Institute, New York, U. S. A. (to Professor G. Asboe-Hansen), Reinholdt W. Jorck og hustrus fond, Kobmand i Odense Johann og Hanne Weimann, f. Seedorff's legat, and The Danish State Research Foundation. The statistical calculations were carried out by Mogens Nyholm, actuary. REFERENCES Boas N. F.: J. biol. Chem. 204 (1953) 553. Braden A. W. H.: Aust. J. sci. Res. B. 5 (1952) 460. Chu J. P.: Endocrinology 34 (1944) 90. Elson L. A. Se Morgan W. T. J.: Biochem. J. 27 (1933) Frederikson H. Se Rydin H.: Acta physiol. scand. 14 (1947) 136. Hofmeister F.: Beitr. klin. Chir. 11 (1894) 441. Jensen C. E. Sc Zachariae F.: Acta endocr. (Kbh.) 27 (1958) 356. Kirk J. E. Se Dyrbye M.: J. Geront. 11 (1956) 273. Krohn P. L.: J. Endocr. 7 (1950) 307. Thorsee H.: Acta endocr. (Kbh.) 37 (1961) 199. Walton A. Se Hammond J.: Brit. J. exp. Biol. 6 (1929) 190. Wislocki G. B., Bunting H. Se Dempsey E. W.: Amer. J. Anat. 81 (1947) 1. Zachariae F.: Acta endocr. (Kbh.) 27 (1958) 339. Zachariae F. Se Jensen C. E.: Acta endocr. (Kbh.) 27 (1958) 343. Received on March 17th, 1962.