0022-1554/92/$3.30

Vol. 40, No. 11. pp. 1705-1713, 1992 Printed in U5.A.

The Journal of Histochemistry and Cytochemistry Copyright 0 1992 by The Histochemical Society, Inc.

Original Article

I Immunohistochemical Localization of Superoxide Dismutases in Fetal and Neonatal Rat Tissues' AFREEN MUNIM, KOHTARO ASAYAMA,2 KAZUSHIGE DOBASHI, KOICHI SUZUKI, AKIRA KAWAOI, and KIYOHIKO KATO Departments of Pediatrics (AM,KA,KD,KK) and Pathology (KS,AK), Yamanashi Medical College, Emaho, Yamanashi 409-38, Japan. Received for publication December 23, 1991 and in revised form June 9, 1992; accepted June 13, 1992 (lA2545).

We investigated the developmental profde of copper-zinc and manganese superoxide dismutase (CuZnSOD and MnSOD) in tissue sections obtained from fetal (Day 12 to 21 of gestation)and neonatal (Day 0 and 6)cats. T h e s were stained iaununohistochemically with specif% antisera against the respective rat SODs. There was a general trend towards richness of SODS in the epithelial linings and metabolically active sites, although differential distribution between the two SODsalso existed. At Day 12 of gestation, immunoreactivity for both SODs was detected in the cardiomyocytes but not in other tissues. Hepatocytes expressedCuznSOD at Day 14 and MnSOD at Day 17. By Day 18 CuZnSOD was detected in the epithelial cells of the gastrointestinaltract, respiratory tract, pancreaticislets, kidneys, and adrenals. These

Introduction Superoxidedismutase (SOD; EC 1.15.1.1) is considered the key enzyme that protects cells against oxidative injury. In animal cells, two forms of intracellular SODs exist. One contains copper and zinc (CuZnSOD) and is found uniformly throughout the nucleus and cytoplasm. The other contains manganese (MnSOD) and occurs predominantly in the mitochondrial matrix (Slot et al., 1986). Levels of lung antioxidant enzymes have been reported to be lower in fetus than in full-term neonate (Hayashibe et al., 1990; Gerdin et al., 1985; Enswell and Freeman, 1984; Yoshioka et al., 1980; Yam et al., 1978). This is assumed to reflect a low demand of cellular antioxidant capacity in fetus due to the low tissue oxygen concentration during intrauterine life (Autor et al., 1976). The abrupt rise in the tissue oxygen concentration after birth facilitates oxidative metabolism, resulting in the increased production of superoxide anion radicals (Freeman and Crapo, 1981). Superoxide can

Supported by Grant-in-Aid #02670431 from the Ministry of Education, Science and Culture of Japan. Correspondence to: Kohtaro Asayama, MD, Dept. of Pediatrics, Yamanashi Medical College, 1110 Shimokato, Emahocho, Nakakomagun, Yamanashi 409-38, Japan.

tissues exhibited MnSOD staining at Day 19. CuZnSOD occurred in the epithelia of the thyroid, thymus, and salivary

glands at Day 19, while MnSOD was seen at Day 21. The increase in intensity of the staining for SODs occurred no later than postnatal Day 0, indicating that most tissues accumulated SODs during late gestation. Breathing atmospheric oxygen during early extrauterine life did not appreciably intensify the SOD staining. These results suggest that perinatal increase in SODs occurs as a general mechanism of preparation for birth. (1&t&em Cytdem 40:1705-1713, 1992) KEY WORDS: Superoxide dismutase; Histochemistry: Free radicals; Oxygen; Fetal development; Rat.

be an initiator of free radical chain reactions, thereby mediating oxidative injury. Accordingly, the inadequate antioxidant protection in premature neonates is postulated to be a primary factor for the pathogenesis of bronchopulmonary dysplasia (Frank and Sosenko, 1987). Reactive oxygen species are reported to cause damage to heart (Simpson and Lucchesi, 1987), kidney (Paller et al., 1984). small intestine (Parks et al., 1983), and various other organs. Apart from fetal lung, information about SOD levels in other fetal tissues is limited. The SOD levels have previously been investigated in fetal rat liver (Pittschieler et al., 1991; Yoshioka et al., 1982; Mavelli et al., 1981), brain (Mariucci et al., 1990), small intestine (Engelhardt et al., 1987), and in heart and kidney (Asayama et al., 1991; Hayashibe et al., 1990). To measure the SOD, biological activity assays have been applied to crude tissue homogenates in some previous studies, and the data are relatively nonspecific and conflicting compared with the data obtained by immunoassay (Asayama et al., 1989). To our knowledge, fetal rat immunoreactive SODs have been investigated only in the lung, kidney, liver, and heart (Pittschieler et al., 1991; Hayashibe et al., 1990). Little is known about the developmental profile of SODS in other tissues. Immunoenzyme staining revealed a wide range of variability in the expression of CuZnSOD and MnSOD from cell to cell (Asayama et al., 1991; Dobashi et al., 1991). Reactive oxygen species are assumed to cause injury at the site of production because 1705

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on March 25, 2015

1706

MUNIM, ASAYAMA, DOBASHI, SUZUKI, KAWAOI, KAT0

A B

of their high reactivity and limited diffusibility, indicating the need for elucidating site-specific changes in SOD. Along these lines of evidence, we investigated the developmental profile of SOD distributions during the perinatal period. Immunohistochemical localization of both CuZnSOD and MnSOD was performed in various rat tissues sequentially from mid-gestation to the early neonatal period.

Materials and Methods Antisera Antisera were raised by immunizing Ncw Zealand White rabbits with purified SODSas described previously (Asayama and Burr. 1985). The spccificity of the antisera has been established by the development of the radioimmunoassay system (Asayama and Burr, 1985) and was also confirmed by immunoblot analysis. Immunoblotting. Rat liver homogenate was subjected to polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate (SDSPAGE). The SDS-PAGEwas performed according to the method of Laemmli (1970). using slab gels (9 x 8 cm) consisting of a 4.5% stacking gel (pH 6.8) and a 10% running gel (pH 8.8) The proteins on the gel were electrophoretically transferred (Towbin et al., 1979) to a nitrocellulose membrane (Atto Corp; Tokyo, Japan) using 25 mM Trisl192 mM glycine containing 10% methanol (pH 8.3) as a blot buffer. After treatment with blocking buffer (15 mM sodium azidell% gclatinlPBS) for 30 min. the membrane was incubated with PBS containing 1% gelatin and antiserum (1:2000 for both CuZnSOD and MnSOD) for 2 hr at 25°C. After washing with 0.3% Tween ZOIPBS. the membrane was incubated with 0.05% antirabbit goat IgG F(ab) fraction labeled with horseradish peroxidase (HRP) (MBL CO;Nagoya, Japan) for 30 min. The HRP activity was visualized by the diaminobenzidine (DAB) reaction (Yokota, 1990). Tissues. Breeding was accomplished by placing male and female Sprague-Dawley rats (Japan SLC; Shizuoka,Japan) together overnight.The midpoint of the cohabitation period was considered to be the onset of pregnancy. Premature pups were delivered by hysterotomy with the dam under pentobarbital anesthesia (50 mglkg) at Days 12. 13, 14, 15, 16. 17, 18. 19, 20. and 21 of gestation, and were decapitated immediately after the delivery. Organs were obtained from the 1- and 7-day-old neonatal rats under ether anesthesia. Two to four litters were sacrificed at each timepoint of the study. Tissues were fixed with 10% buffered formalin, dehydrated through a graded series of ethanol, and embedded in paraffin. Specimens were then cut into 2-pm thicknesses and stained by immunohistochemical technique. Immunostaining. The method for immunostaining of both SODs has been described previously (Dobashi et al., 1989). In brief, after deparaffinization and rehydration the tissue sections were cxposed to 3% H202 for 10 min to inactivate endogenous peroxidase activity and then to 10% normal goat serum for 30 min to block nonspecific binding. The sectionswere incubated with diluted antisera (1:10,000 for anti-CuZnSOD and 1:5000 for anti-MnSOD)overnight at 4’C. then sequentially with 1% anti-rabbit IgG F(ab) fraction labeled with HRP for 60 min. and 0.56 mM DAB11.47 mM H202 for 5 min. Finally, the sections were mounted with Permount (Fisher Scientific; Pittsburgh, PA). As controls, tissue sections were treated in the same way except that antisera were replaced by normal rabbit serum at the same dilutions (Dobashi et al., 1989).

Results Immunoblot AnaZysis Figure 1 shows the data of immunoblot analysisfor rat CuZnSOD

29.0 20.1 14.2 -

6.;-

Figure 1. lmmunoblot analysis of CuZnSOD and MnSOD in adult rat liver homogenate. Molecular weights of the subunits were calibrated with an MW-SDS70L kit (Sigma; St Louis, MO) containing carbonic anhydrase (29.0KO). trypsin inhibitor (20.1 KD),and a-lactalbumin (14.2 KD).(Lane A) 0.5 ul of So/, (whr) liver homogenate, electrophoresedand immunostained with anti-CuZnSOD;(Lane 6) 1.5 VI of the liver homogenate, electrophoresed and immunostained with anti-MnSOD.

and MnSOD in adult rat liver homogenate. Each antiserum depicted a single band corresponding to the subunit of the respective SOD. There was no extraneous substance that crossreacted with the antisera. The molecular masses of the subunits for the CuZnSOD and MnSOD were 17 KD and 22 KD, respectively.

Immunostaining f o r SODs Heart, Lung, and Thymus.Positive staining for both CuZnSOD (Figure 2a) and MnSOD was detected at fetal Day 12 in the cardiomyocytes. but not in other structures of the whole body sections (Tables 1 and 2). Intensity of the staining for CuZnSOD, but not for MnSOD, increased in the cardiomyocytes at Day 21 (Figures 2b and 2c). The bronchiolar epithelium acquired moderate CuZnSOD staining at Day 16 and moderate MnSOD staining at Day 18. The staining for SODs became moderate in the alveolar epithelium (Figure 2d) 1 or 2 days later than in the bronchiolar epithelium. The staining in both epithelia increased in intensity during late gestation (Figures 2e and 2f). The thymus exhibited moderate staining for CuZnSOD at Day 19 and for MnSOD at Day 21. Gastrointestinal Tract. At Day 19 the salivary glands were composed of intercalated ducts, excretory ducts, and terminal tubules with proacinar cells at the distal portion. Both the proacinar cells and ducts were moderately stained for CuZnSOD (Figure 3a). The acinotubular structure developed at Day 21. The ducts were then

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on March 25, 2015

FETAL DEVELOPMENT OF RAT SUPEROXIDE DISMUTASES

1707

Table 1. Immunolocalization of CuZnSOD in fetal rat tissues PIX- md postnatal days' Tissues

12

13

14

15

16

17

18

19

20

21

0

+6

Cardiomyocytes Lungs Bronchiolar epithelium Alveolar epithelium Thymus Salivary glands Acinar cells Ducts Stomach Columnar cells Parietal cells Intestines (small) Columnar cells Goblet cells Hepatocytes Kidneys Glomerulus Undifferentiated tubules Proximal tubules Loop of Henle Distal tubules Collecting ducts Thyroid cells Pancreas Islets Acinar cells Adrenals Fetal cortex Adult cortex Medulla

+

+

+

+

+

+

+

+

+

++

++

++

* * *

*

* *

-

+

+ f

+ +

+ + +

++ ++ +

++ ++

++

f

++

+

+

++ ++ +

* *

*

+ ++

++

* * * * * * * * * *

* * *

*

* *

f

*

* * f

*

* * * *

*

*

*

f

*

* * +

* * *

*

*

* * *

*

*

*

* *

*

*

f

*

-

* +

*

*

f

f

*

+ +

+

-

-

+

-

+

++

f

f

*

+ *

+ *

+

++ +

++

f

+

+

+

+

+

++ + +

++

+

++ + +

-

-

-

-

-

-

+

+

+

+

++ + + + +

++ +

*

-

*

+

* +

*

-

-

-

f

f

* * * *

f

* *

*

*

* *

*

*

f

*

*

+ +

*

*

*

+

-

f

f

+

*

*

*

*

-

+ -

+

+

-

f

f

+

*

* *

* * *

-

-

*

*

f

+

-

-

* *

*

*

f

* +

+ * +

*

+

*

f

+ + +

+

+ +

++ +

+ * +

+ + +

+ +

+

+ ++ +

+ +

+ + +

+

++ +

+ + +

+ +

+

,+

a + + , intensely stained; + , moderately stained: i , equivocally stained, difficult to distinguish from background; - , clearly unstained; *, tissues not yet dcvclopcd. Fetal adrenal cortex had involuted by postnatd Day 6.

stained intensely and the acinar cells moderately for both SODs (Figures 3b and 3c). From Day 13 to Day 17, CuZnSOD was expressed on the surface of the gastric epithelium (Figure 3d). The columnar cells were moderately stained for both CuZnSOD (Figure 3e) and MnSOD at Day 18. At Day 21 the epithelium protruded into the lumen, forming gastric pits. The parietal cells could be recognized at this stage. The columnar cells were stained intensely for CuZnSOD and moderately for MnSOD (Figures 3f and 3g). Conversely, the parietal cells were stained intensely for MnSOD and moderately for CuZnSOD. The primitive villi of the intestinal epithelium were stained moderately for both MnSOD (Figure 3h) and CuZnSOD from Day 18. From Day 21, the epithelial cells were stained intensely, and the goblet cells moderately, for both MnSOD (Figure 3i) and CuZnSOD. The hepatocytes displayed moderate staining for CuZnSOD from Day 14 (Figure 4a). The staining in the hematopoieticcells increased in intensity at Day 18, whereas that in the hepatocytes was unaltered (Figure 4b). Both the hepatocytes and hematopoietic cells were moderately stained for MnSOD from Day 17 (Figure 4c), and intensity of the staining was unaltered thereafter. Kidneys. Undifferentiated tubules could be recognized from

Day 15. They did not acquire moderate staining. At Day 18 the tubules differentiated into the proximal and distal tubules. The former exhibited moderate staining for CuZnSOD (Figure 4d). At Day 19 the proximal and distal tubules and the collecting ducts were moderately stained for both SODs. The CuZnSOD staining increased in intensity in the proximal tubules by postnatal Day 0 (Figure 4e), while the MnSOD staining was intensified in the distal tubules and collecting ducts by Day 21 (Figure 4f). The glomeruli were not stained. Endocrine Glands. CuZnSOD occurred at Day 19 and MnSOD at Day 21 in the follicular epithelium of the thyroid (Figures 5a and 5b). Intensity of the staining for CuZnSOD remained unchanged (Figure 5c), but that for MnSOD decreased transiently at birth (Figure Id). Pancreatic islets could be recognized at Day 16. At Day 18 the CuZnSOD staining was more intense in the islets than in the exocrine cells (Figure le). Conversely, the acinar cells were more intensely stained for MnSOD than the islets (Fig ure 5f). Intensityof the staining for CuZnSOD, but not for MnSOD, in the islets increased at Day 21. The medulla and the fetal cortex could be recognized in the adrenalsat Day 15. They acquiredmoderate staining for CuZnSOD (Figure 5g) and MnSOD at Day 18. The adult cortex developed at Day 21 and stained moderately for both

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on March 25, 2015

1708

MUNIM, ASAYAMA, DOBASHI, SUZUKI, KAWAOI, KAT0

Table 2 . Immunolocalization of MnSOD in fetal rat tissues Pre- and postnatal days" 17

18

19

20

21

0

+6

+

+

+

+

+

+

+

f

+

+

f

+ +

+ +

f

f

+

++ + +

++ + +

++ + +

+

+ ++

+ ++

+ ++

*

+ *

+ ++

+

+

++

++

+

+

++ +

+

++ + +

+

++ + +

-

-

-

-

-

+

+

+

+ + ++

+

Tissue8

12

13

14

Cardiomyocytes Lungs Bronchiolar epithelium Alveolar epithelium Thymus Salivary glands Acinar cells Ducts Stomach Columnar cells Parietal cells Intestines (small) Columnar cells Goblet cells Hepatocytes Kidneys Glomerulus Undifferentiated tubules Proximal tubules Loop of Henle Distal tubules Collecting ducts Thyroid cells Pancreas Iselets Acinar cells Adrenals Fetal cortex Adult cortex Medulla

+

+

+

* * *

* * *

* * *

* *

* *

*

*

-

-

+

+

-

-

+

* *

-

*

-

-

+ *

+

* * *

* *

* *

f

+

+

f

+

+

+

*

*

-

-

*

*

+

+

* * * * * *

15

*

16

*

*

*

* * * * *

* * * *

+

*

* *

*

* +

* * *

* *

f

* *

*

*

* *

* *

*

* *

*

*

+

*

*

Discussion No previous study has dealt with the two distinct forms of intracellular SODs in fetal rat tissues as early as Day 12 of gestation. Utilization of the specific immunoenzyme staining method facilitated the study of the variety of tissues in the mid-gestational period of rat fetuses. The present study revealed that the two SODs occurred in the fetal rat tissues shortly after the completion of organogenesis. CuZnSOD was detected earlier than MnSOD in the following organs: lung, thymus, salivary gland, liver, and thyroid. Differential distribution between CuZnSOD and MnSOD, as was previously found in adult rat tissues (Dobashi et al., 1989), was established during late gestation in the columnar cells and parietal cells of the stomach, the islets and acinar cells of the pancreas, the cortex and medulla of the adrenal glands, and in the renal tubules and ducts. Therefore, the expressions of CuZnSOD and MnSOD appeared to be regulated independently of each other in the individual tissues from prenatal development.

*

+

*

+

+

+

+ +

+

+

+

++ ++ +

+ -

*

+

+

+

+

+

+

+

+

+

+

+

*

+ + , intensely stained; + , moderately stained; k , equivocally stained, difficult to distinguish from background; Fetal adrenal cortex had involuted by postnatal Day 6.

SODs (Figures Sh and Si). The fetal cortex was intensely stained for MnSOD at Day 21, but involuted after birth. The adult cortex had acquired intense MnSOD staining by postnatal Day 6.

*

*

+

*

+

+

++ ++ +

+

+ -

*

+

+

+

++ + +

++ + +

++ +

- , clearly unstained; *,

++

tissues not yet developed.

Both SODs were found earliest in the cardiomyocytes and second earliest in the hepatocytes among the tissues studied. This appears to be consistent with the fact that structural and functional development occurs earlier in heart and liver than in the rest of the organs. Maintenance of circulation by the heart is evident early in embryonic development. Rat cardiomyocytes are known to start contracting regularly from Day 9 of gestation (Sissman, 1970). Fetal liver is also known to actively synthesize proteins and enzymes from early gestation (Jones and Rolph, 1985). MnSOD was reported to occur postnatally in rat liver when activity assayswere used for the studies (Mariucciet al., 1990; Mavelli et al., 1981). On the other hand, immunoreactivity was detected previously at Day 18 (Pittschieler et al., 1991). The expression of MnSOD in fetal rat liver was detected 1 day earlier in the present study than in Pittschieler's study, suggesting that the present immunohistochemical staining system is very sensitive. The activity assays generally used are known to be less sensitive than immunoassays. To enhance sensitivity of the assay, the latter is performed at an alkaline pH (around 10). The alkaline pH is well known to enhance the activity of CuZnSOD but not of MnSOD (Forman and Fridovich, 1973), thereby reducing the contribution of MnSOD to the total SOD activity. Furthermore, we previously demonstrated

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on March 25, 2015

FETAL DEVELOPMENT OF RAT SUPEROXIDE DISMUTASES

1709

.

. * . . .

I

.. . . . .. ,

. _-

A

. .

..

: .i'

.a

. F

.

.-'.

Figure 2. Immunohistochemical localization of CuZnSOD and MnSOD in heart and lung. (a) Heart stained for CuZnSOD, prenatal Day 12; (b) heart CuZnSOD. postnatal Day 0; (c) heart MnSOD, postnatal Day 0; (d) lung CuZnSOD, Day 18; (e) lung CuZnSOD, postnatal Day 0; (1) lung MnSOD, postnatal Day 0. TB, terminal bronchioles; A, alveoli. Original magnification x 200. Bars = 50 pm.

that the MnSOD level was lower in rat tissues than in those of human or mouse even when measured by immunoassay (Asayama et al.. 1985). The failure to detect MnSOD activity in the fetal rat liver homogenates in the aforementioned studies may be at least partly due to the alkaline p H used in their assay systems and/or to the presumed relative scarceness of MnSOD compared with the CuZnSOD in the samples. We previously demonstrated that there was a general trend toward richness of either type of SOD in the surface epithelia and also in the metabolically active sites of the adult rats (Dobashi et al, 1989). The present results indicate that similar distribution of SODS is established in most tissues during the late gestational period. In the present parietal cells of the stomach. abrupt occurrence of the intense MnSOD staining was concomitant with the reported onset of active acid secretion (Hervatin et al., 1987). sug-

gesting that the functional maturation of energy-requiring metabolism necessitated the presence of abundant MnSOD in the mitochondria of these cells. Evidence suggests that SODSare distributed preferentially in the sites that are vulnerable to attack by reactive oxygen species (Asayama et al., 1991; Dobashi et al., 1991). In addition to bronchopulmonary dysplasia, necrotizing enterocolitis (Parks et al., 1983) and acute tubular necrosis of premature neonates (Dauber et al., 1976) are also postulated to be mediated by reactive oxygen species. Rapid accumulation of SODSin the intestinal columnar epithelium and renal proximal tubules during late gestation, as observed here, supports the view that these sites are vulnerable to prenatal oxidative injury because of immaturity of the antioxidant capacity. It has been demonstrated that exposure to atmospheric oxygen in vitro is toxic to early-stage embryos and that this toxicity is al-

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on March 25, 2015

1710

MUNIM. ASAYAMA. DOBASHI, SUZUKI, KAWAOI, KAT0

.. . .*...,. . I : .

I

r

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on March 25, 2015

-*a; ,

1.

'

,

i

.'

FETAL DEVELOPMENT OF RAT SUPEROXIDE DISMUTASES

1711

,:.

n

. c

PT

. I

.

._.

"

. .

I

- .

.

.

.:.',

. . .

.. ,-.. 9" ,

.

, .

..

,

,. .

. . . . . .

&..

. . . .

...

6 .............

. . .

I

I-

:

. -.. . .. ..- . .. . .;. .,,-......

.

., . . .

:'

..-

j . . . . . . .. , C . . .

Figure 4. Immunohistochemical localization of SODS in liver and kidney. (a) Liver CuZnSOD, Day 14; (b) liver CuZnSOD, Day 18; (c) liver MnSOD, Day 17; (e) kidney CuZnSOD, Day 18; (t) kidney CuZnSOD, postnatal Day 0; (9) kidney MnSOD, postnatal Day 0. CV, central vein; G, glomerulus; FT, proximal tubules; U, distal tubules; CD, collecting ducts. Original magnifications: a-c x 400; d-f x 200. Bars: a-c = 25 wm; d-f = 50 pm,

leviated in the presence of exogenous SOD in the culture medium (Noda et al., 1991). Therefore, SOD expression appears to be prerequisite for animal cells to acquire protection against oxygen exposure. In the present study, the late gestational increase in intensity of the staining for both CuZnSOD and MnSOD was not confined to lung and kidney but was observed in various other tissues. Assuming that the late gestational increase in SODSobserved in the variety of tissues implies the acquisition of protection against

oxygen, this phenomenon can be ascribed to a general mechanism of preparing for the increase in oxidative stress during extrauterine life. Lung SOD has been reported to be induced by hyperoxia (Hass et al., 1989). Initiation of breathing after birth is known to increase tissue oxygen concentrations from the intrauterine levels. However, breathing atmospheric oxygen during the 6 postnatal days did not appreciably intensify the SOD staining in the present rat tissues.

Figure 3. Immunohistochemical localization of SODS in gastrointestinal tract. (a) Salivary gland CuZnSOD. Day 19; (b) salivary gland CuZnSOD, postnatal Day 0; (c) salivary gland MnSOD. postnatal Day 0; (d) stomach CuZnSOD, Day 14 (lumen at the top); (e) stomach CuZnSOD, Day 18; (f) stomach CuZnSOD, postnatal Day 0; (9) stomach MnSOD, postnatal Day 0; (h) intestine MnSOD, Day 18, (I)intestine MnSOD, neonate. ID, intercalated ducts; ML, muscular layer; LMM, laminar muscularis mucosa; PC. parietal cells; GC, goblet cells. Original magnifications: a-c,e-I x 200; d x 400. Bars: a-c,e-I = 50 pm; d = 25 pm.

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on March 25, 2015

Figure 5. Immunohistochemical localization of SODSin endocrine glands. (a) Thyroid CuZnSOD, Day 19; (b) thyroid MnSOD, Day 21; (c) thyroid CuZnSOD, postnatal Day 0; (d) thyroid MnSOD, postnatal Day 0; (e) pancreas CuZnSOD, Day 18; (1) pancreas MnSOD, Day 18; (9) adrenal CuZnSOD, Day 19: (h) adrenal CuZnSOD, postnatal Day 0; (I) adrenal MnSOD, postnatal Day 0. P, parathyroid gland; I, islets; C. cortex; M. medulla; FC, fetal cortex; AC, adult cortex. Original magnification x 200. Bars = 50 bm.

1712 Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on March 25, 2015

FETAL DEVELOPMENT OF RAT SUPEROXIDE DISMUTASES

Since the two SODSare assumed to be omnipresent in mature animal cells possessing mitochondria, the lack of staining does not exclude their existence at a low level. Similarly, the lack of intensification of the staining during the postnatal period does not imply that the tissue SOD levels remain absolutely unaltered. However, the present results suggest that the change is more drastic in the late gestational period than in the early postnatal period.

Literature Cited Asayama K, Burr IM (1985): Rat superoxide dismutase: purification, labeling, immunoassay and tissue concentration. J Biol Chem 260:2212 Asayama K. Hayashibe H, Dobashi K, Kat0 K (1989): Lipid peroxide and antioxidant enzymes in muscle and nonmuscle of dystrophic mouse. Muscle Nerve 12:742 Asayama K, Hayashibe H, Dobashi K, Uchida N, Kobayashi M, Kawaoi A, Kat0 K (1991): Immunohistochemical study on prenatal development of rat superoxide dismutases in lungs and kidneys. Pediatr Res 29487 Asayama K, Sharp RA, Burr IM (1985): Purification and radioimmunoassays for superoxide dismutases in the mouse. Int J Biochem 17:1171 Autor AP, Frank L, Roberts RJ (1976): Developmental characteristics of pulmonary superoxide dismutase: relationship to idiopathic respiratory distress syndrome. Pediatr Res 10:154 Dauber IM, Krauss AN, Symchych PS, Auld PAM (1976): Renal failure following perinatal anoxia. J Pediatr 88:851

1713

ment of antioxidant enzymes in rat lung, kidney, and heart: marked increase in immunoreactive superoxide dismutases, glutathione peroxidase, and catalase in the kidney. Pediatr Res 27472 Hervatin F, Moreau E, Ducroc R, Garzon B, Avril P, Millet P, Geloso JP (1987): Ontogeny of rat gastric H-K-ATPaseactivity. Am J Physiol242:G28 Jones CT, Rolph TP (9185): Metabolism during fetal life: A functional assessment of metabolic development. Physiol Rev 65:357 Laemmli UK (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680 Mariucci G, Ambrosini MV, Colarieti L, Bruschelli G (1990): Differential changes in Cu, Zn and Mn superoxide dismutase activity in developing rat brain and liver. Experientia 46:753 Mavelli I, Autuori F. Dini L, Spinedi A, CirioloMR, Rotilio G (1981): Correlation between superoxide dismutase, glutathione peroxidase and catalaze in isolated rat hepatocytes during fetal development. Biochem Biophys Res Commun 102:911 Noda Y, Matsumoto H, Umaoka Y, Tatsumi K, KishiJ (1991): Involvement of superoxide radicals in the mouse two-cell block. Mol Reprod Dev 28:356 Paller MS, Hoidal JR, Ferris TF (1984): Oxygen free radicals in ischemic acute renal failure in the rat. J Clin Invest 74:1156 Parks DA, Bulkely GB, Granger DN (1983): Role of oxygen-derived free radicals in digestive tract diseases. Surgery 94:415 Pittschieler K, Lebenthal E, Bujanover Y, PetellJK (1991): Levels of Cu-Zn and Mn superoxide dismutases in rat liver during development. Gastroenterology 100:1062

Dobashi K, Asayama K, Hayashibe H, Uchida N, Kobayashi M, Kawaoi A, Kat0 K (1991): Effect of diabetes mellitus induced by streptozotocin on renal superoxide dismutases in rat: immunoreactivity and histochemical study. Virchows Arch [B] 60:67

Simpson FT,Lucchesi BR (1987): Free radicals and myocardial ischemia and reperfusion injury. J Lab Clin Med 110:13 Sissman NJ (1970): Developmental landmarks in cardiac morphogenesis: comparative chronology. Am J Cardiol 25:141

Dobashi K, Asayama K, Kat0 K, Kobayashi M, Kawaoi A (1989): Immunohistochemical localization of copper-zinc and manganese superoxide dismutase in rat tissues. Acta Histochem Cytochem 22:351

S l o t p , Geuze HJ, Freeman BA, Crapo JD (1986): Intracellular localization of the copper-zinc and manganese superoxide dismutases in the liver parenchymal cells. Lab Invest 55:363

Engelhardt EL, BeggsJC, Neu J (1987): Maturation of antioxidant enzymes in rat small intestine: lack of glucocorticoid stimulation. J Pediatr 111:459

Towbin H, Staehelin T, Gordon J (1979): Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulosesheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350

Forman HJ. Fridovich I(1973): Superoxidedismutase: a comparison of rate constants. Arch Biochem Biophys 158:396 Frank L, Sosenko IRS (1987): Prenatal developmentof lung antioxidant enzymes in four species. J Pediatr 110:106 Freeman BA, CrapoJD (1981): Hyperoxia increases oxygen radical production in rat lung mitochondria. J Biol Chem 256:10986 Gerdin E, Tyden 0, Eriksson UJ (1985): The development of antioxidant enzymatic defense in the prenatal rat lung: activitiesof superoxide dismutase, glutathione peroxidase and catalase. Pediatr Res 19:687 Hass MA, Iqbal J, Clerch LB, Frank L, Massaro D (1989): Rat lung Cu, Zn superoxide dismutase: isolation and sequence of a full-length cDNA and studies of enzyme induction. J Clin Invest 83:1241 Hayashibe H, Asayama K, Dobashi K, Kat0 K (1990): Prenatal develop-

Tanswell AK, Freeman BA (1984): Pulmonary antioxidant enzyme maturation in the fetal rat. I. Developmental profile. Pediatr Res 18:584 Yam J, Frank L, Roberts RJ (1978): Age-related development of pulmonary antioxidant enzymes in the rat. Proc SOCExp Biol Med 157:293 Yokota S (1990): Cytochemicaland immunocytochemicalstudy on the peroxisomes of rat liver after administration of a hypolipidemic drug, MLM-160. Eur J Cell Biol 53:112 Yoshioka T,Shimada T, Sekiba K (1980): Lipid peroxidation and antioxidants in the rat lung during development. Biol Neonate 38:161 Yoshioka T, Ikkehara Y, Shimatani M, Abe K, Utsumi K (1982): Lipid peroxidation and antioxidantsin the rat liver during development. TohokuJ Exp Med 137:391

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on March 25, 2015

Immunohistochemical localization of superoxide dismutases in fetal and neonatal rat tissues.

We investigated the developmental profile of copper-zinc and manganese superoxide dismutase (CuZnSOD and MnSOD) in tissue sections obtained from fetal...
14MB Sizes 0 Downloads 0 Views