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Electron Microscopic Observation of Vallate Taste Buds of Zinc-Deficient Rats with Taste Disturbance
Auris· Nasus· Larynx (Tokyo) 13 (Supp\. I), S 25-S 31, 1986
ELECTRON MICROSCOPIC OBSERVATION OF VALLATE TASTE BUDS OF ZINC-DEFICIENT RATS WITH TASTE DISTURBANCE Takashi KOBAYASHI, M. D. and Hiroshi TOMITA, M. D. Department of Otorhinolaryngology, Nihon University School of Medicine, Tokyo 173, Japan
The influence of zinc deficiency on the sense of taste was assessed experimentally in rats. Growing rats were kept on a zinc-deficient diet to induce a zinc-deficient stage. The two-bottle preference test was used to determine whether taste disturbance might occur in zinc-deficient rats or not, and we observed morphological changes in taste buds of taste rats that showed taste disturbance. Vallate taste buds were used as specimens, and they were perfused with paraformaldehyde-glutaraldehyde and osmium tetroxide, dehydrated with ethyl alcohol and embedded in Epon. The specimens were observed under a JEM lOo-ex eiectronmicroscope. Ultrastructural changes were observed in the taste pores and bud cells of the rats that showed taste disturbance in the zinc-deficient stage. From these results, the influence of zinc deficiency on the sense of taste in the taste pores and bud cells was confirmed electron microscopically by experiments on rats. Taste disturbance is one of the complaints encountered in the Department of Otolaryngology. Some taste disturbances are caused by otitis media chronica, acoustic tumors and facial palsy, but in many cases it is not clearly known why taste disturbances occur. In many patients who complain of taste disturbance, the causes are not known, but we often Presented before 8th International Symposium on Olfaction and Taste Melbourne Australia, August, 1983 Received for Publication November 10, 1985 Request reprints to: Dr. T. Kobayashi, Department of Otorhinolaryngology, Nihon University School of Medicine, 30-1, Oyaguchi-Kamimachi, Itabashi-ku, Tokyo 173, Japan
observe a decrease in serum zinc (TOMITA, 1975). It is reported that the use of zinc sulfate tablets has normalized the serum zinc level and improved taste disturbance (TOMITA, 1977). Zinc is one of the essential trace elements and plays an important role in the human body. Human serum zinc deficienzy is well known to cause diminution of growth and dermatological disorders. However, the precise relationship between zinc deficiency and taste disturbance is not yet clearly understood. HENKIN et al. (1971) reported that taste buds of patients who complained of taste disturbance showed degeneration of bud cells. we prepared artificially zinc-deficient rats by feeding a zinc-deficient diet and used the twobottle preference test to determine whether taste disturbance had occurred or not (RICHTER, 1939). The purpose of this work is 1) to observe the morphological changes in the taste buds or in the bud cells in zinc-deficient rats with taste disturbance and 2) to survey the correlation between taste disturbance and serum zinc deficiency.
Materials and Methods Twenty-five young male Wi star strain rats (4 weeks old, weighing 70-90 g) were used. They were divided into two groups: the RC (control diet rats) and RD (zinc-deficient diet rats) groups. Five rats (RC group) were given a zinc supplemented control diet (33.25 ppm zinc) and 20 rats (RD group) were given
T. KOBAYASHI and H . TOMITA
S 26
Table 1. Zn-deficient diet. 200.0 (g/kg) 650.0 (g/kg) 100 . 0 (g/kg) 2.0 (g/kg) 10.6 (g/kg) 16.5 (g/kg) 1.0 (g/kg) 1.2 (g/kg) 5.0 (g/kg) 0 .8 (g/kg) 1.6 (g/kg) 12.5 (g/kg) 20,000.0 (IV/kg) 3,000.0 (IV/kg) 10.0 (mg/kg) 30.0 (mg/kg) 16.0 (mg/kg) 16.0 (mg/kg) 16 .0 (mg/kg) 40 .0 (mg/kg)
Egg albumin Sucrose Arachis oil Methionine CaC0 3 CaHP0 4 ·2H zO MgCO, MgSO. ·7H 20 NaCI KCI FePO. (soluble) KH 2P0 4 Vit. A Vit.D Menadione a -Tocopheryl acetate Thiamine HCI Riboflavin Pyridoxine HCI Ca pantothenate
Control diet : Zn-deficient diet with Zn (ZnCO, 60.0 mg/kg). Table 2.
Method.
Wi star strain rat (4 weeks old, male)
t
Fixation
I t
1) 2 % paraformaldehyde2.5 % glutaraldehyde 2) 2% osmium tetroxide
Dehydration in ethyl alcohol
t
Embedding in Epon 812
t
Ultrathin sectioning
t
1
Double staining in
1) uranyl acetate 2) lead citrate
The concentrations of quinine hydrochloride used in this experiment were 10- 7 , 10- 6 and 10- 5 M . We concluded from the results of this study that taste-disturbed animals confused quinine solution and drank more of it than distilled water. We compared the volume of distilled water and that of the quinine solution consumed by the rats in 72 hr. Vallate taste buds were used as specimens and were observed by TEM. All the specimens were perfused with a 2 % paraformaldehyde-2.5 %glutaraldehyde solution, followed by postfixation for I hr in 2 % osmium tetroxide, then dehydrated with ethyl alcohol, embedded in Epon 812 and finally stained with uranyl acetate and lead citrate. The specimens were examined under a JEM 100-CX electronmicroscope (Table 2). Results 1) Body weight and diminution of growth. At the start of experiments, the mean weight of the RC and RD rats was 80 g and 78 g, respectively, which was not a significant difference . But after 1 week, the weight of the RC group had increased satisfactorily, while that of the RD group had not increased. The RC group was about 15 g heavier than the RD group. After 7 weeks, the RC group was about 110 g heavier than the RD group (Fig. 1). 2) Dermatological disorders. After 6 or 7
Electronmicroscope (lEM l00-CX)
a zinc-deficient diet (1.96 ppm) (Table I). Rats were kept in environmental control system EBAC-S (manufactured by Japan Kurea Corp.) for a period of 10-15 weeks. A two-bottle preference test was employed to determine whether taste disturbance occurred or not. One bottle contained only plain distilled water and the other a quinine hydrochloride solution. Just before the experiment, the rats were given quinine solution to examine the taste threshold of each rat.
Contr ol diet rats
O~--7--7--~~--~~--~---
t0
2
3
4
5
6
7
start of experiment
Fig. 1. Body weight changes in rats.
week) (
TASTE BUDS OF ZINC-DEFICIENT RATS
S 27
5o,------.....--11--------
8 o
D
=0
S
~IOO~------~~------------------
.3
x
u
o Control diet rats Zinc-deficient diet rats
c:
x
N
g 50~--------------------------'"
VJ
x x
x
O~~1-2~3~4~5~6-7~8~9~ILO~II~I~2-1~3~14-1~5~16~1~7 I start of experiment
(week)
Fig. 2. Serum zinc changes in control diet rats and zinc-deficient diet rats. ( ml )
Fig. 4. Longitudinal section of taste bud (normal rat). Dense substance is observed in the taste pore region and dark granules are observed in the cytoplasm. An arrow indicates taste pore region . x9,000. DS, dense substance; MY, microvilli; DG, dark granule.
25 20
15 10
5
ot
2
3
4
start of experiment
5
6
7
8
9
10 11 12 (week)
Fig. 3. Water consumption in 72 hr : a zinc-deficient rat with taste disturbance. 0 , distilled water; *, quinine solution 10- 6 M.
weeks, depilation, skin disorders resembling acrodermatitis enteropathica and parakeratosis were observed in the RD group. 3) Serum zinc changes. At the start of experiments, the serum zinc of RC and RD rats was indicated to be about lOO ,ug/dl. Through the experiment, the RC group's serum zinc was 100-150,ug/dl, but in the RD group the serum zinc decreased rapidly over 7 weeks (Fig. 2). 4) Two-bottle preference test. RC group rats showed a good distinction between quinine solution and distilled water. But in the RD group, the consumption of quinine
Fig. 5. Taste buds of zinc-deficient rats with taste disturbance. An arrow indicates loss of dense substance in the taste pore region. x 5,000.
solution increased in about 4 weeks and was greater than distilled water consumption in 8 weeks. Finally 9 out of 20 rats tended to
S 28
T. KOBAYASHI and H. TOMITA
Fig. 6. Taste buds of zinc-deficient rats with taste disturbance. Microvilli are broken down and decreased in number. An arrow indicates loss of dense substance in the taste pore region. No hyperkeratosis of the epithelium or blockage of taste pore region is observed. x 32,000. Co Y, cored vesicle; MY, microvilli; DG, dark granule.
consume more quinine solution than distilled water and showed taste disturbance (Fig. 3). 5) Ultrastructural changes of taste pores and bud cells (Figs. 4-9). 1. Dense substance was lost in the taste pore region. 2. Microvilli decreased in number as compared with those of normal animals and were partly broken down. 3. Dense granules could be observed but they decreased in number in the type I cells. 4. Cytoplasmic microvacuolation in the bud cells was clearly demonstrated. 5. Clear vesicles in the pointed end of the microvilli, which are not usually found in the normal structure, were observed in these rats, and in the type I cells organelles were seen as an abnormal structure. 6. The mitochondrial matrix and cristae degenerated in the type I cells. 7. Cristae of the Golgi apparatus enlarged extraordinarily and they showed lysosomal characteristics.
Fig. 7. Taste buds of zinc-deficient rats with taste disturbance. Dark granules decrease in the cytoplasm. Microvilli decrease in number and are broken down. An arrow indicates loss of dense substance in the taste pore region and double arrows clearly indicate cytoplasmic microvacuolation in the bud cells. x 13,000. MY, microvilli; DG, dark granule.
8. Rough surfaced endoplasmic reticuli expanded in part. 9. In the multivesicular bodies, granules degenerated and the matrix showed lysosomal characteristics. 10. Fibrils increased in number in the cytoplasm. Discussion In many patients who complain of taste disturbance, it is reported that their serum zinc is decreased and by using zinc sulfate tablets internally, serum zinc is normalized and taste disturbance is improved. On the other hand, in growing rats, the minimum necessary concentration of zinc is
TASTE BUDS OF ZINC-DEFICIENT RATS
Fig. 8. Taste buds of zinc-deficient rats with taste disturbance. Vesicles are found in the pointed end of microvilli in the pore region. x 40,000. CI V, clear vesicle; MV, microvilli.
S 29
reported to be about 10-18 ppm, but the zincdeficient diet that we used contained 1.96 ppm, which is very low for the growth of rats (PALLAUF, 1972; CATALANOTTO, 1977). The serum zinc of the RC group was within 100150 ,ug/dl, but that of the RD group was below 50 ,ug/dl. HASEGAWA et al. (1980) reported that even if rats indicated a marked zinc deficiency, only 15 % of them showed taste disturbance. Therefore in this experiment, we used the two-bottle preference test to distinguish taste disturbed rats from rats with normal taste. In our experiment, 9 out of 20 rats showed taste disturbance. In these 9 rats, we observed bud cells with TEM. Vallate taste buds look like oval bodies and are opened to the gap. The outer taste pore can be seen as an opening in the flattened keratinized epithelial cells. The hole is called the outer taste pore and ends in the inner taste pore (OGES, 1976). The inner
Fig. 9. Taste buds of zinc-deficient rats with taste disturbance. Rough-surfaced endoplasmic reticuli expand in part. Cristae of the Golgi apparatus are extraordinarily enlarged and show lysosomal characteristics. Mitochondrial matrix and cristae degenerate in type I cells. In the multivesicular bodies, granules degenerate and the matrix shows lysosomal characteristics. Fibrils increase in number in the cytoplasm. x 17,000. Go, Golgi apparatus; Mit, mitochondria; N, nucleus; MB, multivesicular body; F, fibril; rER, rough surfaced endoplasmic reticuli.
S 30
T. KOBAYASHI and H. TOMITA
taste pore is constituted from a part of the granules protects the microvilli from stimulabud cells and microvilli, and an electron dense tion. substance and pore vesicles can be seen in it. Zinc deficiency might induce the producBud cells are distinguished into 3 types tion of dark granules from the bud cells and (ADVIDSON, 1976). decrease the dense substance among microMicrovilli with diameters of from 0.2- villi. From these results, there appears to 0.4 mm are in the normal range, but they be a connection between the decrease in the decreased in number and were partly broken protective function of microvilli and the loss down in taste disturbed rats. The dense sub- of microvilli. stance among the microvilli is histochemically polysaccharide and is seen as a uniform, high Summary density substance in TEM. Considering that HENKIN et al. (1975) reported that there is the dense substance came from the dark a loss of the dense substance in the taste pore granules of type 1 cells and the dark granules region, a loss of microvilli and a decrease of in type 1 cells had decreased remarkably, the dark granules and microvacuolation in the loss of the dense substance among the micro- taste pore buds of patients who complain of villi is caused by underproduction and un- taste disturbance. In our experiment, we dersecretion of dark granules from type 1 prepared artificially zinc-deficient rats by feedcells (SHIMAMURA, 1972; MURRAY, 1973). ing a zinc-deficient diet and observed the same Dark granules are produced in the Golgi ap- results in the taste buds of zinc-deficient rats paratus of type 1 cells, and we observed that with taste disturbance. the cristae of the Golgi apparatus were extraordinarily enlarged. They showed lysoReferences somal characteristics in the taste disturbed ADVTDSON, K.: Scanning electron microscopy of animals and caused underproduction of the fungiform papillae in the tongue of man and dark granules in the Golgi apparatus of type monkey. Acta Otolaryngol. 81: 496-502, 1976. CATALANOTTO, F. A., and NANDA, R.: The effects of 1 cells. feeding a zinc deficient diet on taste acuity and The taste buds of the rats in a zinc-deficient tongue epithelium in rats. l. Oral Pathol. 6: 211condition showed many abnormal changes in 220,1977. TEM. These changes were thought to be im- CiGES, M.: Ultrastructural study of taste buds at rest portant for the stimulation of the sense of and after stimulation and comparative study between type 3 cell and merkel cells. Acta Ototaste. A taste transmitting substance is neceslaryngol. 81: 209-219, 1976. sary to stimulate the bud cells of the sense of taste. In the first stage, the transmitting sub- HASEGAWA, H., TOMITA, H., and KISHI, T.: Abnormal taste reactivities of zinc deficient rats. Prostances from the gap have to reach and ceedings of Japanese Association for the Study of stimulate the microvilli in the pore region. Taste and Smell, Vol. 14, pp. 69-71, 1980 Without this process, the transmitting sub- HENKIN, R. I., SCHECHTER, P. J., and HOYE, R. H.: Idiopathic hypogeusia with dysgeusia, hyposmia stance cannot stimulate the bud cells. What and dysosmia. A new syndrome. lAMA. 217: is the role of the dense substance? The dark 434-440, 1971. granules are a precursor substance of the dense HENKIN, R. I., PATTEN, B. M., and REP. K.: A new substance among microvilli. The dense subsyndrome of acute zinc loss. Arch. Neurol. 32: stance is continuously excreted from the taste 745-751, 1975. pore region to the papilla gap. Microvilli, MURRAY, R. G.: The ultrastructure of taste buds. In The Ultrastructure of Sensory Organs, pp. 1-81, which have receptor sites for taste transmitFriedmann American Elsevier, North-Holland, ters, are continuously exposed to many heavy 1973. stimulations from the pore region, and we PALLAUF, J.: Zinkgehalte in Knochen und Ganzbelieve that the dense substance from the dark korper wachsender Ratten bei unterschiedlichere
TASTE BUDS OF ZINC-DEFICIENT RATS Zinversorgung. Zum Stoffwechsel des Zinks im tierschen Organismus. Z. Tierphysiol. 30: 193202, 1972. RICHTER, C. P.: Salt taste threshold of normal and adrenalectomized rats. Am. J. Physiol. 24: 367371, 1939. SHIMAMURA, A.: Scanning electron microscopic observations on the taste pores and taste hairs in rabbit gustatory papillae. Arch. Histol. Jpn . 34 :
S 31
51-60, 1972. TOMITA, H.: Zinc and taste disturbance. Proceedings of Trace Metal Metabolism, Vol. 1, pp. 61-68, 1975. TOMITA, H .: Taste disturbance due to zinc deficiency and its treatment. Proceedings of Japanese Association for the Study of Taste and Smell, Vol. 11, pp. 40-47, 1977.
ELECTRON MICROSCOPIC OBSERVATION OF VALLATE TASTE BUDS OF ZINC-DEFICIENT RATS WITH TASTE DISTURBANCE Takashi KOBAYASHI, M. D. and Hiroshi TOMITA, M. D. Department of Otorhinolaryngology, Nihon University School of Medicine, Tokyo 173, Japan
The influence of zinc deficiency on the sense of taste was assessed experimentally in rats. Growing rats were kept on a zinc-deficient diet to induce a zinc-deficient stage. The two-bottle preference test was used to determine whether taste disturbance might occur in zinc-deficient rats or not, and we observed morphological changes in taste buds of taste rats that showed taste disturbance. Vallate taste buds were used as specimens, and they were perfused with paraformaldehyde-glutaraldehyde and osmium tetroxide, dehydrated with ethyl alcohol and embedded in Epon. The specimens were observed under a JEM lOo-ex eiectronmicroscope. Ultrastructural changes were observed in the taste pores and bud cells of the rats that showed taste disturbance in the zinc-deficient stage. From these results, the influence of zinc deficiency on the sense of taste in the taste pores and bud cells was confirmed electron microscopically by experiments on rats. Taste disturbance is one of the complaints encountered in the Department of Otolaryngology. Some taste disturbances are caused by otitis media chronica, acoustic tumors and facial palsy, but in many cases it is not clearly known why taste disturbances occur. In many patients who complain of taste disturbance, the causes are not known, but we often Presented before 8th International Symposium on Olfaction and Taste Melbourne Australia, August, 1983 Received for Publication November 10, 1985 Request reprints to: Dr. T. Kobayashi, Department of Otorhinolaryngology, Nihon University School of Medicine, 30-1, Oyaguchi-Kamimachi, Itabashi-ku, Tokyo 173, Japan
observe a decrease in serum zinc (TOMITA, 1975). It is reported that the use of zinc sulfate tablets has normalized the serum zinc level and improved taste disturbance (TOMITA, 1977). Zinc is one of the essential trace elements and plays an important role in the human body. Human serum zinc deficienzy is well known to cause diminution of growth and dermatological disorders. However, the precise relationship between zinc deficiency and taste disturbance is not yet clearly understood. HENKIN et al. (1971) reported that taste buds of patients who complained of taste disturbance showed degeneration of bud cells. we prepared artificially zinc-deficient rats by feeding a zinc-deficient diet and used the twobottle preference test to determine whether taste disturbance had occurred or not (RICHTER, 1939). The purpose of this work is 1) to observe the morphological changes in the taste buds or in the bud cells in zinc-deficient rats with taste disturbance and 2) to survey the correlation between taste disturbance and serum zinc deficiency.
Materials and Methods Twenty-five young male Wi star strain rats (4 weeks old, weighing 70-90 g) were used. They were divided into two groups: the RC (control diet rats) and RD (zinc-deficient diet rats) groups. Five rats (RC group) were given a zinc supplemented control diet (33.25 ppm zinc) and 20 rats (RD group) were given
T. KOBAYASHI and H . TOMITA
S 26
Table 1. Zn-deficient diet. 200.0 (g/kg) 650.0 (g/kg) 100 . 0 (g/kg) 2.0 (g/kg) 10.6 (g/kg) 16.5 (g/kg) 1.0 (g/kg) 1.2 (g/kg) 5.0 (g/kg) 0 .8 (g/kg) 1.6 (g/kg) 12.5 (g/kg) 20,000.0 (IV/kg) 3,000.0 (IV/kg) 10.0 (mg/kg) 30.0 (mg/kg) 16.0 (mg/kg) 16.0 (mg/kg) 16 .0 (mg/kg) 40 .0 (mg/kg)
Egg albumin Sucrose Arachis oil Methionine CaC0 3 CaHP0 4 ·2H zO MgCO, MgSO. ·7H 20 NaCI KCI FePO. (soluble) KH 2P0 4 Vit. A Vit.D Menadione a -Tocopheryl acetate Thiamine HCI Riboflavin Pyridoxine HCI Ca pantothenate
Control diet : Zn-deficient diet with Zn (ZnCO, 60.0 mg/kg). Table 2.
Method.
Wi star strain rat (4 weeks old, male)
t
Fixation
I t
1) 2 % paraformaldehyde2.5 % glutaraldehyde 2) 2% osmium tetroxide
Dehydration in ethyl alcohol
t
Embedding in Epon 812
t
Ultrathin sectioning
t
1
Double staining in
1) uranyl acetate 2) lead citrate
The concentrations of quinine hydrochloride used in this experiment were 10- 7 , 10- 6 and 10- 5 M . We concluded from the results of this study that taste-disturbed animals confused quinine solution and drank more of it than distilled water. We compared the volume of distilled water and that of the quinine solution consumed by the rats in 72 hr. Vallate taste buds were used as specimens and were observed by TEM. All the specimens were perfused with a 2 % paraformaldehyde-2.5 %glutaraldehyde solution, followed by postfixation for I hr in 2 % osmium tetroxide, then dehydrated with ethyl alcohol, embedded in Epon 812 and finally stained with uranyl acetate and lead citrate. The specimens were examined under a JEM 100-CX electronmicroscope (Table 2). Results 1) Body weight and diminution of growth. At the start of experiments, the mean weight of the RC and RD rats was 80 g and 78 g, respectively, which was not a significant difference . But after 1 week, the weight of the RC group had increased satisfactorily, while that of the RD group had not increased. The RC group was about 15 g heavier than the RD group. After 7 weeks, the RC group was about 110 g heavier than the RD group (Fig. 1). 2) Dermatological disorders. After 6 or 7
Electronmicroscope (lEM l00-CX)
a zinc-deficient diet (1.96 ppm) (Table I). Rats were kept in environmental control system EBAC-S (manufactured by Japan Kurea Corp.) for a period of 10-15 weeks. A two-bottle preference test was employed to determine whether taste disturbance occurred or not. One bottle contained only plain distilled water and the other a quinine hydrochloride solution. Just before the experiment, the rats were given quinine solution to examine the taste threshold of each rat.
Contr ol diet rats
O~--7--7--~~--~~--~---
t0
2
3
4
5
6
7
start of experiment
Fig. 1. Body weight changes in rats.
week) (
TASTE BUDS OF ZINC-DEFICIENT RATS
S 27
5o,------.....--11--------
8 o
D
=0
S
~IOO~------~~------------------
.3
x
u
o Control diet rats Zinc-deficient diet rats
c:
x
N
g 50~--------------------------'"
VJ
x x
x
O~~1-2~3~4~5~6-7~8~9~ILO~II~I~2-1~3~14-1~5~16~1~7 I start of experiment
(week)
Fig. 2. Serum zinc changes in control diet rats and zinc-deficient diet rats. ( ml )
Fig. 4. Longitudinal section of taste bud (normal rat). Dense substance is observed in the taste pore region and dark granules are observed in the cytoplasm. An arrow indicates taste pore region . x9,000. DS, dense substance; MY, microvilli; DG, dark granule.
25 20
15 10
5
ot
2
3
4
start of experiment
5
6
7
8
9
10 11 12 (week)
Fig. 3. Water consumption in 72 hr : a zinc-deficient rat with taste disturbance. 0 , distilled water; *, quinine solution 10- 6 M.
weeks, depilation, skin disorders resembling acrodermatitis enteropathica and parakeratosis were observed in the RD group. 3) Serum zinc changes. At the start of experiments, the serum zinc of RC and RD rats was indicated to be about lOO ,ug/dl. Through the experiment, the RC group's serum zinc was 100-150,ug/dl, but in the RD group the serum zinc decreased rapidly over 7 weeks (Fig. 2). 4) Two-bottle preference test. RC group rats showed a good distinction between quinine solution and distilled water. But in the RD group, the consumption of quinine
Fig. 5. Taste buds of zinc-deficient rats with taste disturbance. An arrow indicates loss of dense substance in the taste pore region. x 5,000.
solution increased in about 4 weeks and was greater than distilled water consumption in 8 weeks. Finally 9 out of 20 rats tended to
S 28
T. KOBAYASHI and H. TOMITA
Fig. 6. Taste buds of zinc-deficient rats with taste disturbance. Microvilli are broken down and decreased in number. An arrow indicates loss of dense substance in the taste pore region. No hyperkeratosis of the epithelium or blockage of taste pore region is observed. x 32,000. Co Y, cored vesicle; MY, microvilli; DG, dark granule.
consume more quinine solution than distilled water and showed taste disturbance (Fig. 3). 5) Ultrastructural changes of taste pores and bud cells (Figs. 4-9). 1. Dense substance was lost in the taste pore region. 2. Microvilli decreased in number as compared with those of normal animals and were partly broken down. 3. Dense granules could be observed but they decreased in number in the type I cells. 4. Cytoplasmic microvacuolation in the bud cells was clearly demonstrated. 5. Clear vesicles in the pointed end of the microvilli, which are not usually found in the normal structure, were observed in these rats, and in the type I cells organelles were seen as an abnormal structure. 6. The mitochondrial matrix and cristae degenerated in the type I cells. 7. Cristae of the Golgi apparatus enlarged extraordinarily and they showed lysosomal characteristics.
Fig. 7. Taste buds of zinc-deficient rats with taste disturbance. Dark granules decrease in the cytoplasm. Microvilli decrease in number and are broken down. An arrow indicates loss of dense substance in the taste pore region and double arrows clearly indicate cytoplasmic microvacuolation in the bud cells. x 13,000. MY, microvilli; DG, dark granule.
8. Rough surfaced endoplasmic reticuli expanded in part. 9. In the multivesicular bodies, granules degenerated and the matrix showed lysosomal characteristics. 10. Fibrils increased in number in the cytoplasm. Discussion In many patients who complain of taste disturbance, it is reported that their serum zinc is decreased and by using zinc sulfate tablets internally, serum zinc is normalized and taste disturbance is improved. On the other hand, in growing rats, the minimum necessary concentration of zinc is
TASTE BUDS OF ZINC-DEFICIENT RATS
Fig. 8. Taste buds of zinc-deficient rats with taste disturbance. Vesicles are found in the pointed end of microvilli in the pore region. x 40,000. CI V, clear vesicle; MV, microvilli.
S 29
reported to be about 10-18 ppm, but the zincdeficient diet that we used contained 1.96 ppm, which is very low for the growth of rats (PALLAUF, 1972; CATALANOTTO, 1977). The serum zinc of the RC group was within 100150 ,ug/dl, but that of the RD group was below 50 ,ug/dl. HASEGAWA et al. (1980) reported that even if rats indicated a marked zinc deficiency, only 15 % of them showed taste disturbance. Therefore in this experiment, we used the two-bottle preference test to distinguish taste disturbed rats from rats with normal taste. In our experiment, 9 out of 20 rats showed taste disturbance. In these 9 rats, we observed bud cells with TEM. Vallate taste buds look like oval bodies and are opened to the gap. The outer taste pore can be seen as an opening in the flattened keratinized epithelial cells. The hole is called the outer taste pore and ends in the inner taste pore (OGES, 1976). The inner
Fig. 9. Taste buds of zinc-deficient rats with taste disturbance. Rough-surfaced endoplasmic reticuli expand in part. Cristae of the Golgi apparatus are extraordinarily enlarged and show lysosomal characteristics. Mitochondrial matrix and cristae degenerate in type I cells. In the multivesicular bodies, granules degenerate and the matrix shows lysosomal characteristics. Fibrils increase in number in the cytoplasm. x 17,000. Go, Golgi apparatus; Mit, mitochondria; N, nucleus; MB, multivesicular body; F, fibril; rER, rough surfaced endoplasmic reticuli.
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T. KOBAYASHI and H. TOMITA
taste pore is constituted from a part of the granules protects the microvilli from stimulabud cells and microvilli, and an electron dense tion. substance and pore vesicles can be seen in it. Zinc deficiency might induce the producBud cells are distinguished into 3 types tion of dark granules from the bud cells and (ADVIDSON, 1976). decrease the dense substance among microMicrovilli with diameters of from 0.2- villi. From these results, there appears to 0.4 mm are in the normal range, but they be a connection between the decrease in the decreased in number and were partly broken protective function of microvilli and the loss down in taste disturbed rats. The dense sub- of microvilli. stance among the microvilli is histochemically polysaccharide and is seen as a uniform, high Summary density substance in TEM. Considering that HENKIN et al. (1975) reported that there is the dense substance came from the dark a loss of the dense substance in the taste pore granules of type 1 cells and the dark granules region, a loss of microvilli and a decrease of in type 1 cells had decreased remarkably, the dark granules and microvacuolation in the loss of the dense substance among the micro- taste pore buds of patients who complain of villi is caused by underproduction and un- taste disturbance. In our experiment, we dersecretion of dark granules from type 1 prepared artificially zinc-deficient rats by feedcells (SHIMAMURA, 1972; MURRAY, 1973). ing a zinc-deficient diet and observed the same Dark granules are produced in the Golgi ap- results in the taste buds of zinc-deficient rats paratus of type 1 cells, and we observed that with taste disturbance. the cristae of the Golgi apparatus were extraordinarily enlarged. They showed lysoReferences somal characteristics in the taste disturbed ADVTDSON, K.: Scanning electron microscopy of animals and caused underproduction of the fungiform papillae in the tongue of man and dark granules in the Golgi apparatus of type monkey. Acta Otolaryngol. 81: 496-502, 1976. CATALANOTTO, F. A., and NANDA, R.: The effects of 1 cells. feeding a zinc deficient diet on taste acuity and The taste buds of the rats in a zinc-deficient tongue epithelium in rats. l. Oral Pathol. 6: 211condition showed many abnormal changes in 220,1977. TEM. These changes were thought to be im- CiGES, M.: Ultrastructural study of taste buds at rest portant for the stimulation of the sense of and after stimulation and comparative study between type 3 cell and merkel cells. Acta Ototaste. A taste transmitting substance is neceslaryngol. 81: 209-219, 1976. sary to stimulate the bud cells of the sense of taste. In the first stage, the transmitting sub- HASEGAWA, H., TOMITA, H., and KISHI, T.: Abnormal taste reactivities of zinc deficient rats. Prostances from the gap have to reach and ceedings of Japanese Association for the Study of stimulate the microvilli in the pore region. Taste and Smell, Vol. 14, pp. 69-71, 1980 Without this process, the transmitting sub- HENKIN, R. I., SCHECHTER, P. J., and HOYE, R. H.: Idiopathic hypogeusia with dysgeusia, hyposmia stance cannot stimulate the bud cells. What and dysosmia. A new syndrome. lAMA. 217: is the role of the dense substance? The dark 434-440, 1971. granules are a precursor substance of the dense HENKIN, R. I., PATTEN, B. M., and REP. K.: A new substance among microvilli. The dense subsyndrome of acute zinc loss. Arch. Neurol. 32: stance is continuously excreted from the taste 745-751, 1975. pore region to the papilla gap. Microvilli, MURRAY, R. G.: The ultrastructure of taste buds. In The Ultrastructure of Sensory Organs, pp. 1-81, which have receptor sites for taste transmitFriedmann American Elsevier, North-Holland, ters, are continuously exposed to many heavy 1973. stimulations from the pore region, and we PALLAUF, J.: Zinkgehalte in Knochen und Ganzbelieve that the dense substance from the dark korper wachsender Ratten bei unterschiedlichere
TASTE BUDS OF ZINC-DEFICIENT RATS Zinversorgung. Zum Stoffwechsel des Zinks im tierschen Organismus. Z. Tierphysiol. 30: 193202, 1972. RICHTER, C. P.: Salt taste threshold of normal and adrenalectomized rats. Am. J. Physiol. 24: 367371, 1939. SHIMAMURA, A.: Scanning electron microscopic observations on the taste pores and taste hairs in rabbit gustatory papillae. Arch. Histol. Jpn . 34 :
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51-60, 1972. TOMITA, H.: Zinc and taste disturbance. Proceedings of Trace Metal Metabolism, Vol. 1, pp. 61-68, 1975. TOMITA, H .: Taste disturbance due to zinc deficiency and its treatment. Proceedings of Japanese Association for the Study of Taste and Smell, Vol. 11, pp. 40-47, 1977.