The effect of hypophysectomy in the weanling rat on size of the submandibular gland and on salivary proteins

The effect of hypophysectomy in the weanling rat on size of the submandibular gland and on salivary proteins

A&s oralBiol.Vo1.12, pp.721-731, 1967. Pergmon Press Ltd.Printed inGt.Britain. THE EFFECT OF HYPOPHYSECTOMY IN THE WEANLING RAT ON SIZE OF THE SUBMAN...

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A&s oralBiol.Vo1.12, pp.721-731, 1967. Pergmon Press Ltd.Printed inGt.Britain.

THE EFFECT OF HYPOPHYSECTOMY IN THE WEANLING RAT ON SIZE OF THE SUBMANDIBULAR GLAND AND ON SALIVARY PROTEINS D. F. CARBONE, E. A. SWEENN and J. H. SHAW Harvard School of Dental Medicine, Boston, Massachusetts,

U.S.A.

Summary-Hypophysectomy in the weanling rat resulted in major reductions in the rate of growth, in food consumption and in both the absolute and relative weights of endocrine target organs in comparison with intact rats fed a semi-purified diet adlibitum. The ratios of the weight of the submandibular gland complex to body weight in male and female rats were similar for hypophysectomized and normal rats, which suggests that the gross size of the submandibular gland was not uniquely dependent on the secretion of pituitary hormones. Pilocarpine-stimulated salivary secretion and the total amount of salivary protein secreted were closely related to body weight and to submandibular gland weight in normal and hypophysectomized rats; salivary protein concentration tended to be elevated in the hypophysectomized rats. During paper electrophoresis, altered distributions of the protein components of saliva were observed in the experimental animals with notable increases in the anodal migrators. These changes in saliva were comparable to previously described alterations in hypothyroid rats. INTRODUCTION IN RECENT years evidence from animal experiments has indicated that endocrine function and salivary gland morphology are closely interrelated and has led to the hypothesis that possible changes in the quality and quantity of saliva may be partly responsible for the differences in caries experience observed during various endocrinopathies (SHAW, 1950; MTJHLER and SHAFER, 1952: SHAFER and MIJHL.ER, 1954; MIJHLER, BIXLER and SHAFER, 1956; HALDI, WYNN and LAW, 1962; SHAFER and MIJHLER, 1963; CARBONE, SWEENEY and SHAW, 1966). The pituitary-salivary gland relationship in the rat has received considerable attention. Hypophysectomy resulted in increased experimental dental caries (SHAFER and MIJHLER, 1955); in the submaxillary gland investigators have observed decreased proteolytic activity (SREEBNY, 1953), decreased amylolytic activity (SHAFERand MUHLER, 1955), atrophy of granular tubules (SHAFERand MUHLER, 1955) and depletion of acinar ribonucleic acid (RNA) (BIXLER et al., 1957: KRONMAN and CHAUNCEY, 1964). The administration of appropriate hormones to hypophysectomized rats has been shown to result in the restoration to normal of the microarchitecture, the enzyme activities and localization, and the RNA content of the salivary glands (SHAFER, CLARK and MIJHLER, 1956; BIXLER,MUHLER and SHAFER,1959). Agreement has not been reached concerning any biologic mechanisms which might explain differences in caries incidence during endocrine imbalance. Even though saliva has been shown to be an essential component of the relationship 721

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0.

F. CARBONE, E. A. SWEENEY AND J. H. SHAW

between thyroid function and incidence of dental caries (HALDI,WYNNand LAW, 1962), no consistent alteration in salivary gland function has been related to caries experience in endocrinopathies in general. SWEENEY et al. (1962) described a method of paper electrophoretic separation and characterization of the protein components of pilocarpine-stimulated whole rat saliva. With this method, CARBONE,SWEENEYand SHAW (1966) demonstrated consistent differences in the salivary protein composition and caries experience of hyper- and hypothyroid rats. In part, these associations between salivary proteins and caries experience paralleled similar trends observed by SWEENEYet al. (1962) in intact caries-resistant and susceptible rats. The present experiment was an attempt to ascertain the relationships of hypophysectomy in the weanling rat among size of submandibular glands, distribution of salivary proteins, food consumption and rate of body growth.

EXPERIMENTAL METHOD Albino rats of the Charles River strain were divided at 21 days of age by sex and litter into control Group A with twenty-two rats and experimental Group B with twenty-eight rats. The same day animals in Group B were hypophysectomized by way of the parapharyngeal approach. All animals were housed in individual wire bottom cages and supplied with water and purified ration 2700 ad Iibitum (SHAWand GRIFFITHS,1960). Each rat was weighed thrice weekly, and food consumption for representative members of each group was measured daily. At 80 days of age whole saliva was collected as described previously for exactly 7 min after subcutaneous injection of pilocarpine hydrochloride solution at 7.5 mg/kg body weight (CARBONEet al., 1966). The volume of each sample of collected saliva was measured in a pipet graduated in 0.01 ml. Total salivary protein concentrations were determined uniformly for all animals by the method of LOWRYet al. (1951). Blood samples were collected by cardiac puncture and, after clot retraction, were centrifuged at 2500 rev/min for 20 min and sera removed. Paper-electrophoretic separations of serum and salivary proteins were carried out on the day of collection in the manner reported by SWEENEY et al. (1962), with the modification proposed by DAWES(1963). The animals were then sacrificed by excess ether inhalation. Immediately the submandibular gland complex (the submaxillary and the major sublingual glands), thyroid, adrenal and sex organs were dissected free and weighed. At frequent intervals during the experiment, the hypophysectomized male rats had been palpated for gross evidence of increase in testicular size. Two male rats were discarded when testicular enlargement was noted; two female rats died within one week of surgery. The remaining twenty-four hypophysectomized rats survived the 59-day experimental period. Criteria for complete hypophysectomy included decreased rate of growth and decreased food consumption throughout the experiment, and reduced size of the adrenals, thyroids, testes, prostate and ovaries at the termination of the experiment. The consistency of all these values for the twenty-four

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rats remaining at the end of the experiment led to their inclusion in the tabulation of data. Serial sectioning of the sella turcica was considered to be unnecessary and gross examination of the sella less adequate than the composite data listed above. RESULTS

The data in Table 1 on the actual weights of the rats and of their submandibular gland complexes, adrenals, thyroids, prostates, testes or ovaries indicated the profound and generalized growth-restricting effect of hypophysectomy. The reduced tissue weights among hypophysectomized rats in comparison to normal values were the most striking for the prostates, testes or ovaries. The general growth-restricting influence included the complete elimination of the customary difference in body size between male and female rats of the same age. Also in Table 1 are presented data on the tissues expressed as ratios of the actual weights of the organ in mg to 100 g of body weight. When the values for the submandibular gland complex in hypophysectomized rats of either sex were compared to those of normal rats, no significant differences were observed. In contrast, similar comparisons for the adrenals, prostates, testes and ovaries indicated that these organs had been even more restricted in their relative growth after hypophysectomy than body weight. For each of these tissues, these differences from normal were statistically significant (p 0.05
>

All comparisons

11 13

Females Control Hypophysectomized

tp < 0.01 :p > 0.01< 0.05

11 11

Males Control Hypophysectomized

No. of rats

425 172t

624 1761 166 169

153 165

(mg/ (mg) 1OOg)

41.6 7.0t

17.1 7.7t

19.9 8-2

(mg)

6.6 7.5:

4.9 7*7t

(mg/ loo&

Thyroid

-

3090 129t

(mg)

-

760 122t

-

510 07

-

125 07

(mf3/ 1OOg)

Prostate

(mg/ 1OOf3) (mg)

Testes

rats and the controls of the same sex.

16.3 6.9t

34.4 8.5 7.07 6.7:

(mg/ (mg) 1OOf3)

Adrenals

are made between hypophysectomized

257 102t

408 107’i

Body weight (l3)

Submand. gland

TABLE~.~INFLUENCEOFHYPOPHYSECTOMYONAVERAGEFINALBODY~IGHTS,TISSUEWEIGHTSANDRA~OSOFTISSUE~GHTSTOFINALBODY

61.0 4.5t

--

(mg)

24.0 4.4t

-

(mgl 1OOg)

Ovaries

WEIGHTS

HYPOPHYSECTOhIYANDSALIVARYPROTEINS

FIGS. 1 and 2. Relationships between body weight and total food consumption in control and hypophysectomized rats. The solid line represents body weight at weekly intervals; the broken line represents cumulative total food consumption at weekly intervals. The horizontal lines above and below each point indicate the standard error of the mean. The total number of animals represented by each point is shown in parentheses. Data for males are presented in Fig. 1 and for females in Fig. 2. TABLE 2. THE INFLUENCE OF HYPOPHYSECTOMY ON THE AVERAGE TOTAL SALIVARY FLOW, SALIVARY PROTEIN CONCENTRATION AND TOTAL SALIVARY PROTEIN SECRETED

Total flow (ml/7 min) Males Control Hypophysectomized Females Control Hypophysectomized *Determined tp < 0.01

Protein concentration* (mg/ml)

1.06 0.3ot

8.4 10.2:

0.82 0*33t

9.2 IO.0

after the method of LOWRY et al. (1951) $p > 0.01 < 0.05

Total protein secreted (mg/7 min)

Et

726

D. F.

CARBONE, E. A. SWFENEY AND

SHAW

J. H.

mg of submandibular gland complex was slightly higher for hypophyscctomized rats of both sexes than for their normal littermates. The data for the paper electrophoretic analyses of serum proteins are shown in Table 3. Since preliminary evaluation of the data demonstrated no significant sex difference for serum protein components, male and female values in each group were TABLE 3. PAPER ELECTROPHORETIC ANALYSES OF SERUM PROTEINS

‘A of total protein present* Globulins No. of rats Control Hypophysectomized

Albumin

22 23

37 36

*Determined by densitometric fp > 0.01 < 0.05

Alpha,

Alpha,

17 19t

14 13t

Total alpha

Beta

31 32

Gamma

17

14

I@

16t

__

analysis of serum electrophoretograms.

combined and evaluated. Hypophysectomized rats demonstrated decreases of borderline significance (p> 0.01 <0*05) in alpha, and beta globulin fractions and increases of borderline significance in alpha, and gamma globulin fractions. None of these differences was sufficiently large td merit serious consideration. The albumin : globulin (A/G) ratio was similar in both groups. In Table 4 are recorded salivary protein components (expressed as mg %) numbered after the method of CARBONE et al. (1966). The major differences in the saliva of hypophysectomized rats were the highly significant increases of components 1, 2, 3 and 4, the faster anodal migrators. TABLE 4. THE INFLUENCE OF HYPOPHYSECTOMY ON THE CONCENTRATION OF SALIVARY PROTEIN COMPONENTS

Component No. of rats Control Hypophysectomized

13 14

1

2

3

4

39 77t

63 89t

(mg %)* 5

6

7

8

9

271 280

104 95

22 28

60 80:

21 I6

*Estimated by converting densitometric analyses of salivary electrophoretograms to component salivary protein concentrations from total salivary protein concentrations, individually for each animal. tp< 0.01 :p > 0.01 < 0.05 DISCUSSION

Hypophysectomy in the young rat maintained on a semi-purified diet has been shown to be compatible with life and to allow a slow amount of growth in the skeletal and soft tissues, even though voluntary food consumption was drastically reduced (SHAW and GREEP, 1949). The amount of body growth in the current experiment was

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727

very similar to that in this earlier investigation. When the rats were sacrificed after 59 days, the actual weight of the submandibular gland complex among hypophysectomized rats was much less than that of the control rats. However, when the ratios of the submandibular gland complex to body weight were compared between hypophysectomized and normal rats, they were observed to be almost identical. The possibility must be considered that the lesser amount of food consumed by the hypophysectomized rats led to less mastication and to smaller salivary glands. The latter concept grows out of the studies of HALL and SCHNEYER (1964) who observed much smaller parotid, submaxillary and sublingual glands among rats fed a liquid diet than among those fed a standard pelleted ration where both diets were fed ad libitum. They attributed the decrease in gland weights among the rats on the liquid diet to the reduced amount of mastication. They did not test the influence of caloric restriction on gland weights. Our comparison of the combined weights of the submaxillary and sublingual glands with the body weights of HALL and SCHNEYER’S animals fed the solid ration indicated lower ratios than we observed in either our hypophysectomized or normal rats. HALL, MERIGand SCHNEYER (1966) have shown that the alterations in the flow rates of saliva in humans on a liquid diet could be reversed to normal with the substitution of an isocaloric solid diet, thus implicating only the process of mastication. However, in both studies the submaxillary and sublingual glands were only minimally affected in comparison to the parotid and in neither experiment to the extent observed in our experiments. Therefore, the above hypothesis concerning the possible influence of food consumption on salivary gland weight in our animals does not appear to have validity. Further support for this lack of relationship can be obtained from the pair-fed controls in the thyroid experiments of CARBONE et al. (1966), where 12 and 38 per cent caloric restriction did not cause decreased ratios of submandibular gland weight to body weight. This information strongly suggested that the gross physical size of the submandibular glands was not under special endocrine influence from the hypophysis but grew in our hypophysectomized rats at a rate comparable to that of the entire body. This fact was in distinct contrast to the adrenals, prostates, testes and ovaries of the hypophysectomized rats where tissue growth was less rapid relatively than the rate of body growth as judged by the ratio of tissue weight to body weight. For these organs, the absence of the specific adrenocorticotrophic and gonadotrophic hormones were reflected in the hypophysectomized rat by significantly lower ratios of organ weight to body weight than were observed in the normal rat. These data closely parallel those reported in early classical studies such as that of CROOKEand GILMOUR(1938). The thyroid gland appeared to be in a somewhat different category. Though the gross weight of the thyroid glands was greatly reduced in the hypophysectomized rats, the ratio of thyroid weight to body weight among hypophysectomized rats was slightly greater than for normal rats. Hypophysectomy resulted in dramatic reductions in the total salivary flow after pilocarpine stimulations in the 7-min test period. Since salivary secretion expressed on the basis of ml/l00 g of body weight or/100 mg of submandibular gland weight was

728

D. F. CARBONE, E. A. SWEENEY AND J. H. SHAW

comparable for normal and hypophysectomized rats, this decrease in flow appeared to be more closely related to the body weight and submandibular gland weight than to the endocrine status of the rat. This observation was consistent with the finding that the ratios of the weight of the submandibular gland complex to body weight were comparable for normal and hypophysectomized rats. The slight increases in protein concentration of the saliva of hypophysectomized rats was grossly insufficient to increase the total amount of protein secreted during the test period to the level secreted by normal rats. However, on a body weight or salivary gland weight basis, the amount of protein secreted by hypophysectomized rats tended to be slightly higher than for normal rats. Since the crowns of the molar teeth of these rats were almost fully developed at the beginning of the experiment, the size and morphology of the molar crowns could not have been expected to differ appreciably between the two groups. Therefore the lesser amount of saliva secreted in the hypophysectomized rats would have the same area of tooth surface to be distributed over as the much larger amount of saliva in the normal rats. Thus the reduced volume of saliva observed in the hypophysectomized rats could have played a role in the production of the higher incidence of dental caries that has been observed by other workers (SHAFERand MUHLER,1955). In addition, quality differences in the proteins of saliva were observed to have been altered by hypophysectomy and its sequelae. The saliva of hypophysectomized rats exhibited significant increases in anodally migrating components 1, 2, 3 and 4 compared to intact rats. These changes among the hypophysectomized rats were consistent with the grossly visible increased viscosity of the saliva in comparison with that of the control littermates. The increased viscosity may be especially attributable to the increase in component 1 which is mutinous in nature and derived solely from the submandibular gland complex (SWEENEYet al., 1962). Component 2, analogous in electrophoretic mobility to serum albumin, although increased significantly in the saliva of hypophysectomized rats, was not elevated in the serum. Component 3, analgous in mobility to serum alpha globulins, was increased in hypophysectomized rat saliva yet total alpha globulins in hypophysectomized rat serum remained unchanged. Component 4 is unique to saliva, has no serum analogue, and is as yet unidentified. In view of recent observations by MANDEL(1966), these components are more likely small groups of proteins and should not be strictly considered as single molecular species. These changes in distribution and concentration of the proteins in saliva did not parallel the more minor changes observed in the serum proteins. This lack of relationship suggested that the salivary protein components were not wholly dependent upon the composition of the blood, indicating either de now synthesis of proteins in the salivary glands or selective filtration, or more likely both mechanisms. The electrophoretic distribution of salivary proteins in hypophysectomized rats resembled, in many respects, that of saliva secreted by hypothyroid rats (CARBONE et al., 1966). This similarity may be an expression of the hypothyroid condition resulting from the absence of thyrotropin in hypophysectomized rats. In the latter experiment, hypothyroid rats were compared to pair weight control animals whose

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129

rates of growth were limited to those of the hypothyroid group by caloric restriction. Caloric restriction in the intact animal resulted in a decrease in salivary component 5 (analogous in mobility to serum beta globulins, containing salivary amylase) and an increase in component 9 (analogous in mobility to egg white lysozyme). Saliva from hypothyroid rats demonstrated an increase in component 5 and a decrease in component 9 compared to intact calorically restricted rats. When saliva from hypophysectomized rats in the present experiment was compared to saliva from intact animals receiving food ad libitum, no differences were observed in components 5 and 9. If hypophysectomized rats had been compared to intact rats of comparable food intake, then an increase in salivary component 5 and decrease in component 9 in the former might have been manifested. A further refinement of the present experiment would entail the inclusion of a group of normal rats that were pair-fed controls of the hypophysectomized rats to determine whether the above assumption is correct. Interestingly, SWEENEYet al. (1962) have shown that the only differences in salivary protein components between a caries-susceptible and caries-resistant strain of rats were the higher level of 5 and lower level of 9 in the former. Thus in these conditions where caries incidence tends to be elevated, a trend appears to be developing for salivary component 5 to be higher in concentration and component 9 to be lower in concentration than among situations where caries incidence tends to be low. These changes in the composition of saliva from the hypophysectomized rats are undoubtedly closely related to the histological and enzymatic changes in the salivary glands that have been described by other workers (SREEBNY,1953; SHAFERand MUHLER,1955; BIXLERet al., 1957; and KRONMANand CHAUNCEY,1964). Probably these quality changes in saliva may be even more closely related to the increased caries incidence among hypophysectomized rats by SHAFERand M~_~HL,ER (1955). Our studies have not included comparable evaluations of size of the parotid and minor sublingual salivary glands which have contributed to the mixed saliva that we collected and analyzed. The specific role that the changes in the salivary proteins may have played is presently unknown and may continue to be so until exhaustive investigations by isolation and characterization have been undertaken. Acknowledgements-This investigation was supported in part by Public Health Service Fellowship DPO-15,636, Career Award 5-K6-DE22,634 and research grant DE-00373 from the National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland, U.S.A. We are grateful to the Merck, Sharp and Dohme Research Laboratories, West Point, Pennsylvania, for the generous supplies of B-complex vitamins used in the diets in these studies, and to Miss INARAKRUMINSfor her indispensable help in the experimentation. RQum&-Des hypophysectomies de rats sevres provoquent une importante diminution de la croissance, de la consommation alimentaire et du poids absolu et relatif des glandes endocrines, en rapport avec l’hypophyse, lorsqu’on compare ces donntks avec des rats temoins, soumis a volonte a un regime semi-purifie. Les rapports du poids des glandes sous-maxillaires par rapport au poids corporel des rats males et femelles sont identiques pour les rats nonnaux et hypophysectomids, indiquant que

730

D. F.

CARBONE, E.

A.

SWEENEY

ANDJ. H. SHA\\

la taille dc la glande sous-maxillaire n’est pas uniquement Ii&eB la s&r&ion d’hormones hypophysaires. La s&&ion salivaire, stimulee par pilocarpine, ainsi que la quantitt totale de proteine salivaire s&ret&e sont en rapport ttroit avec le poids corporcl ct le poids de la glande sous-maxillaire chez les rats normaux et hypophysectomisCs; la concentration en prot&ine salivaire tend g &tre plus elev&e dans les rats hypophysectom&s. Par ilectrophor&se sur papier, on note une alt6ration de la distribution des composCs proteiques de la salive dans les animaux opCrCs, avec augmentation des fractions anodiques. Ces modifications salivaires sont comparables g celles d&rites chez le rat hypothyroldien. Zusammenfassung-Die Hypophysektomie fiihrte bei der entwahnten Ratte im Vergleich zu intakten Ratten, die ad libitum eine hablgereinigte Dilt erhielten, zu erheblicher Reduktion der Wachstumsrate, der Nahrungsaufnahme und des absoluten wie such des relativen Gewichts der endokrin abhgngigen Organe. Die Verhlltnisse des Gewichts des Submandibulardriisenkomplexes zum Kijrpergewicht war fiir hypophysektomierte und normale Ratten bei Mtinnchen und Weibchen lhnlich; dies legt nahe, daI3 die G&se der Submandibulardriise nicht allein von der Sekretion der Hypophysenhormone abhingt. Die Pilocarpin-stimulierte Speichelsekretion und die Gesamtmenge des sezernierten Speichelproteins stand bei normalen und hypophysektomierten Ratten in naher Beziehung zum Kiirpergewicht und zum Gewicht der Submandibulardriise; bei den hypophysektomierten Ratten neigte die Proteinkonzentration des Speichels zur ErhGhung. Papierelektrophoretisch wurden bei den Versuchstieren verPnderte Verteilungen der Proteinbestandteile des Speichels mit bemerkenswerter Zunahme der anodisch wandernden Substanzen beobachtet. Diese Speichelverlnderungen waren mit friiher beschriebenen Vertinderungen bei hypothyroiden Ratten verglelchbar.

REFERENCES BIXLER,D., MUHLER, J. C. and SHAFER,W. G. 1959. Growth hormone and thyroxine; elfects on slubmaxillary gland of hypophysectomized rat. Proc. Sot. exp. Biol. Med. 100, 400402. BIXLER,D., MUHLER, J. C., WEBSTER,R. D. and SHAFER,W. G. 1957. Changes in submaxillary gland ribonucleic acid following hypophysectomy, thyroidectomy and various hormone treatments. Proc. Sot. exp. Biol. Med. 94, 521-524. CARBONE,D. F. SWEENEY,E. A. and SHAW, J. H. 1966. The comparative influence of thyroid imbalance and limited body weight gain on submandibular gland weight, the protein components of saliva and dental caries in the rat. Archs oral Biol. 11, 781-792. CROOKE,A. C. and GILMOLJR,J. R. 1938. A description of the effect of hypophysectomy on the growing rat with the resulting histological changes in the adrenal and thyroid glands and the testicles. J. Path. Butt. 47, 525-544. DAWES,C. 1963. Disodium ethylenediaminetetraacetatc as an aid for the reconstitution of lyophilized human salivary proteins before paper electrophoresis. Archs oral Biol. 8, 653-656. HALDI, J., WYNN, W. and LAW, M. 1962. Relationship between thyroid function and resistance to dental caries. J. dent. Res. 41, 398404. HALL, H. D., MERIG, J. J. and SCHNEYER,C. A. 1966. Metrecal-induced changes in human salivary glands. International Association for Dental Research, 44th General Meeting, Abstract 186. HALL, H. D. and SCHNEYER,C. A. 1964. Salivary gland atrophy in rat induced by liquid diet. Proc. Sot. exp. Biol. Med. 117, 789-793.

KRONMAN,J. H. and CHAUNCEY,H. H. 1964. Hormonal influence on rat submandibular gland histochemistry. J. dent. Res. 43, 520-527. LOWRY,0. H., ROSEBROUGH, W. J., FARR, A. L. and RANDALL,R. J. 1951. Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 263-275. MANDEL,I. D. 1966. Electrophoretic studies of saliva. J. dent. Res. 45, 634-643. MUHLER,J. C., BIXLER,D. and SHAFER,W. G. 1956. Effect of replacement therapy on dental caries experience of radiothyroidectomized rats. Proc. Sot. exp. Biol. Med. 93, 328-330. MUHLER,J. C. and SHAFER,W. G. 1952. Experimental dental caries. 1. The effects of orchiectomy and ovariectomy on dental caries in immature rats. J. dent. Res. 31,798-804. SHAFER,W. G., CLARK, P. G. and MUHLER, J. C. 1956. The inhibition of hypophysectomy-induced changes in the rat submaxillary glands. Endocrinology 59, 516-521.

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SHAFER,W. G. and MUHLER, J. C. 1954. Experimental dental caries. III. The effect of estradiol and diethylstilbcstrol on dental caries, fluorine metabolism, and the salivary glands of intact and gonadectomized rats. J. dent. Res. 33, 842-848. SHAFER, W. G. and MUHLER, J. C. 1955. Experimental dental caries. IV. The effect of hypophysectomy on dental caries and the salivary glands of the rat. J. dent. Res. 34, 531-536. SHAFER,W. G. and MUHLER, J. C. 1963. Endocrine influences upon the salivary glands. Ann. N. Y.

Acad. Sci., The Metabolism of Oral Tissues 85,215-227. SHAW, J. H. 1950. Studies on the effect of gonadectomy on dental caries activity in white rats and in cotton rats. J. dent. Res. 29, 798-801. SHAW, J. H. and GREEP, R. 0. 1949. Relationships of diet to the duration of survival, body weight and composition of hypophysectomized rats. Endocrinology 44,520-535. SHAW, J. H. and GRIFFITHS,D. 1960. Partial substitution of hexitols for sucrose and dextrin in caries-producing diets. J. dent. Res. 39, 377-384. SREEBNY,L. M. 1953. Histologic and enzymatic differences in the submaxillary glands of normal and hypophysectomized male and female white rats. J. dent. Res. 32,686 (abstract). SWEENEY,E. A., SHAW, J. H., CHILDS, E. L. and WEISBERGER,D. 1962. Studies on the protein composition of rodent saliva. Archs oral Biol. 7, 621-631.

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