Association Between Mitochondria and Rough Endoplasmic Reticulum (rer) in Rat Hepatocytes

Path. Res. Pract. 180, 49-53 (1985)

Association Between Mitochondria and Rough Endoplasmic Reticulum (rer) in Rat Hepatocytes Kanchan Pal Singh*, Heinz David, Thomas v. Zglinicki Department of Electron Microscopy of the Charite, Institute of Pathology of the Humboldt University Berlin, GDR

SUMMARY

The relative contact area between rough endoplasmic reticulum and mitochondria of hepatocytes in newborn to 27 months old rats is described by morphometric methods. The contact area decreases from 52.62% to 27.46% of the mitochondrial surface between newborn and rats being 27 months old. However, there is an increase in the contact area per hepatocytic cytoplasm and in the fraction of rer in contact with mitochondria during the first days post partum.

Introduction The association between various membrane bound organelles has long appealed to biologists. Mietkiewsky18, David and Noconll, Ghidom and Thomas 14, Morrt~ et al,2°, Shore and Tata28 , Pickett et al. 22 , Cascarano et a1. 6 and Wolf and Schimassek33 emphasized the association between rough endoplasmic reticulum and mitochondria. However, there is no systematic morphometric study on this association, which may quantify and clarify its functional significance. This study, therefore, deals with this association in rat liver during postnatal development. Material and Methods The study is based on the liver of male Wistar rats at birth, at 1-7, 14 and 21 days, and at 1-6, 12, 18, 24 and 27 months of age. Three animals of each group were examined. They were bred under conventional conditions - in common plastic pans on a standard diet and given water ad libitum, room temperature (20-22 0c) and relative humidity (70-80%). The animals were checked for normal physical development by subjective assessment of appearance, checking chow consumption and body weight.

* Zoology Department, Agra College, Agra (India) © 1985 by Gustav Fischer Verlag, Stuttgart

Tissue specimens were excised from three regions of the livers of each animal, prefixed in a modified Karnovsky solution (glutaraldehyde 2.5%, paraformaldehyde 1.0%, Cacodylate buffer 0.1 M); dehydrated in graded alcohol solutions and embedded in Micropal. The tissue was sectioned on a LKB Ultrotome and stained with uranyl acetate and lead citrate. One block per region was chosen randomly and examined in a Siemens Elmiskop I a and Elmiskop 102. Six micrographs were taken from one section by a systematic sampling scheme (Weibel 1979, p. 82) at a final magnification of x 20000. The perimeter of the mitochondria LM and the length Ls of the mitochondrial outer membrane surrounded by rer (Fig. 1) were measured with the aid of a semiautomatic tracing device MOP AM02 (KONTRON). All mitochondria on the image were measured, provided they had a clear delineated surface. Construction of histograms and analysis of variance between group means was done on-line on a HP 9825 A desk computer. Because of the random sampling32 it holds per definition: F - ILs _ Sc,v - ILM - SM,V with SM,V and Sc v ... surface density of the outer mitochondrial membrane and the part of it in connection with the rer, respectively, in the containing space (cytoplasm) and ILs and IL M... sums of all measured values of Ls and LM, respectively, per age group. Therefore F is not only a measure in a two-dimensional space (section) but the fraction of the mitochondrial surface in 0344-0338/8510180-0049$3.5010

50 . K. P. Singh, H. David and T. v. Zglinicki

Fig. 1. Hepatocytic cytoplasm of newborn (a), 6 months (b), 1 year (c) and 2:4 year old (d) rats illustrating the decreasing degree of association between rer and mitochondria with age. The bar denotes 1 J.Lm.

connection with the rer or the contact surface fraction. Knowing the data of SM,V and Vrer,V (= volume density of rer in the cytoplasm) of the same rats from David 10 the following secondary parameters were computed:

- the contact surface density in the hepatocytic cytoplasm Sc,v = F· SMV - the co'ntact surface density in relation to the volume of rer, SC,rer

=

SC,V/Vrer,V •

Rer-mitochondrial Association . 51

Results Table 1 and Fig. 2 show that the contact surface fraction is near 50% of the mitochondrial surface in newborn, 1-6 and 7 days old rats. In 3 days old ones like that in two weeks to 1 month old rats it is near 45%. The contact surface fraction decreases up to 42% in 2 months, 36% in 6 months, and 27% in 1 year. In 1liz years it slightly increase (32%), remains almost the same at 2 years stage, and again decreases at 214 years stage to come in accord with 1 year stage. These findings, thus, show but a fluctuating decline in the contact area between the two organelles with increasing age. However, it becomes almost consistent after 1 year stage, with no further decline. Table 2 indicates that most mitochondria are partly surrounded by the portions of rer. In the newborn rats, about 14% of the mitochondrial sections were free of rer, their

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Table 1. Mitochondrial contact surface fraction F, mean perimeter of the mitochondrial sections LM and number N of mitochondria measured per age group

Newborn 1 day 2 days 3 days 4 days 5 days 6 days 7 days 2 weeks 3 weeks 1 month 2 months 3 months 4 months 5 months 6 months 1 year 1.5 year 2 years 2.25 years

LM

F (%) Mean SD

SE

49.49 47.51 50.36 44.35 49.61 53.62 50.44 50.95 44.46 44.85 45.64 42.00 38.39 37.05 34.67 36.51 27.58 32.10 33.85 27.46

1.96 3.00 1.58 3.74 1.93 2.70 1.75 3.10 1.28 3.05 1.27 2.66 1.33 2.87 1.50 2.48 1.29 2.83 1.16 2.64 1.36 2.88 1.48 2.76 1.39 2.57 1.42 2.40 1.52 2.63 1.65 2.58 1.69 2.60 1.43 2.48 1.82 2.75 1.53 2.82

30.09 30.30 30.97 26.22 25.11 23.57 21.49 24.46 22.91 21.32 21.86 25.01 24.18 24.96 24.46 24.89 25.37 26.38 26.56 20.75

Mean SD

N SE

1.00 0.07 1.45 0.07 0.93 0.06 0.99 0.07 0.91 0.05 0.97 0.05 1.08 0.07 0.75 0.05 0.99 0.06 0.86 0.05 0.89 0.06 0.81 0.05 0.71 0.04 0.68 0.04 0.68 0.04 0.69 0.05 0.81 0.05 0.58 0.03 0.67 0.05 0.60 0.04

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Fig. 2. Contact surface density per volume of rer SC,rer (open circles), per hepatocytic cytoplasm Sc,v (crosses) and mitochondrial contact area fraction F (black dots) vs. age. The data for F are given as mean ± standard error. The horizontal lines mark means of groups of only insignificantly different values according to an analysis of variance. Significances between these groups are given by asterisks.

52 . K. P. Singh, H. David and T. v. Zglinicki Table 2. Relative frequencies of the fraction of the perimeter of mitochondrial sections in contact with rer

% of mitochondrial perimeter

Age

0

Newborn 1 day 2 days 3 days 4 days 5 days 6 days 7 days 2 weeks 3 weeks 1 month 2 months 3 months 4 months 5 months 6 months 1 year 1.5 year 2 years 2.25 years

14.8 3.7 17.3 26.2 22.4 12.8 6.5 23.4 24.5 13.8 12.1 5.9 19.9 24.9 15.1 8.9 5.7 32.4 31.1 10.2 6.8 4.5 23.2 33.0 21.6 4.9 3.5 16.5 34.3 29.1 3.1 2.6 23.7 42.7 20.2 4.5 5.2 22.4 34.8 19.0 4.2 11.5 28.2 30.4 17.6 4.4 6.8 30.3 36.5 17.0 3.1 7.8 32.2 33.2 18.2 10.8 8.3 29.7 27.2 17.7 11.0 8.9 40.2 19.3 14.4 13.6 9.4 35.7 24.0 10.1 15.8 9.7 39.2 20.7 9.2 17.2 7.5 33.9 23.7 11.0 32.3 4.9 34.9 17.2 7.6 6.2 33.2 18.3 10.3 27.1 23.6 5.2 34.0 22.6 9.0 21.6 5.9 52.5 14.6 3.3

20

40

60

80

99.9

100

12.2 3.0 9.5 9.7 10.6 11.7 4.4 7.1 6.3 5.7 6.7 4.9 3.5 4.6 7.1 7.1 5.8 3.5 3.0 2.4 3.5 3.1 5.2 3.1 4.4 2.7 3.5 3.9 3.4 1.9 3.6 2.6 0.8 2.7 1.8 3.2 1.4 4.2 2.2

number decreases till the 6th day (3.1%) and increases constantly to become near 32% in 1 year. It again decreases up to 27, 23 and 21 % in 1%, 2 and 214 years stages respectively. In general, the data of Table 3 reflect the decline of the contact area with age. The contact surface density per cytoplasm Sc,v approximately parallels the behaviour of F Table 3. However, there is a quite sharp decrease between 1 and 4 weeks of age, and a clear increase during the first days post partum. This increase is even more pronounced for the contact fraction of the rer as measured by the surface density SC,rer'

Table 3. Age dependence of the contact surface density per hepatocytic volume Sc,v and per volume of rer SC,rer Age

Sc,v (m 2 /cm 3 )

SC,rer (m 2 /cm 3 )

Newborn 1 day 2 days 3 days 4 days 5 days 6 days 7 days 2 weeks 3 weeks 1 month 2 months 3 months 4 months 5 months 6 months

0.59 0.62 0.71 0.68 0.74 0.80 0.72 0.95 0.77 0.71 0.54 0.55 0.54 0.53 0.50 0.52

12.62 15.48 24.54 21.80 25.62 22.58 21.36 21.03 16.43 11.76 15.18 14.13 10.20 7.36 11.70 9.69

Discussion A close contact between rough endoplasmic reticulum and mitochondria has been reported in mouse sciatic nerve24 ; oocytes of hamster31 ; guinea pig1; rabbit34 and mouse2?; rat luteal cells 8, fungi 3, onion stem20 , amphibian liver?, and in rat liver4,6,9,n,12,22,33. However, such a contact between mitochondria and rough endoplasmic reticulum is not readily discernible in adipose tissue, cardiac muscle and skeletal muscle13. There is little information on the quantitative aspect of this association. Picker21 found that 5-7% of the total cellular rough endoplasmic reticulum cosedimented with mitochondria. Montisano et a1. 19 noticed 81 % isolated mitochondria in contact with rough endoplasmic reticulum. Wolf and Schimassek33 found a small fraction of rough endoplasmic reticulum (containing maximally 15% of the cytoplasmic ribonucleic acid) in contact with mitochondria. David and Nocon ll found a contact surface fraction of 0.52 ± 0.19 in mouse hepatocytes. According to this study the mean contact area per mitochondrial surface decreases from about 50% of the mitochondrial surface in newborn rats to about 27% in rats being 214 years old. Significant changes occur between 1 and 2 weeks, 2 and 3 months, 6 and 18 months and 24 and 27 months. So the data may be grouped together in accordance with juvenile, young, adult and old ages. Only the 3 days and 12 months values fall out of this order. The functional significance of the association between these organelles has been differently interpreted. Robertson24 and Andet? supposed its involvement in mitochondrial genesis. Picket et aU2 suggested a possible role of this association in biosynthesis of cytochrome P-450; while Cascarano et al. 6 hypothecated that succinic dehydrogenase is synthesized on the mitochondrially associated rough endoplasmic reticulum in preparation for transfer to the mitochondria. Porter23 and Caro and Palade5 related rough endoplasmic reticulum to the synthesis and intracellular transport of proteins; while Truman30 and Roodyn25,26 reported that the only proteins synthesized by mitochondria are the insoluble structural proteins; and Simpson et aU9, Roodyn et aU5,26 and Kadenbach 16 pointed out that mitochondria are unable to synthesize the soluble proteins (i.e. proteins of the major metabolic pathways). Further, Kadenbach 16 and Halder et alY demonstrated that soluble proteins are synthesized on the cytoplasmic membranes and subsequently transferred to the mitochondria. It seems clear that the association between rer and mitochondria facilitates this transfer. For the discussion of functional significance it may be interesting that (i) the fraction of rer in contact with mitochondria Sc rer increases about twofold within two days after birth together with a sharp increase in mitochondrial volume density and (ii) the contact surface density in cytoplasm decreases between 1 week and 1 month during a strong increase of the number of hepatocytes (Fig. 5 of David, 1985), that means during a period of strong mitotic activity. Thus, during the early postnatal development contact rer is formed parallel to mitochon-

Rer-mitochondrial Association . 53

drial growth, much faster than the total rer. In addition, since this association has been observed in several different cell types, its universal significance calls to be detected. Acknowledgements. The first author is grateful to the authorities of Indo-GDR Cultural Exchange Programme for providing an opportunity to work in GDR; and to Dr. W. Krause for helping throughout the work.

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Received April 24, 1984 . Accepted in revised form July 11, 1984

Key words: Morphometry - Mitochondria - Rough endoplasmic reticulum - Liver - Age dependence Prof. Dr. Heinz David, Dept. of Electron Microscopy of the Charite, Institute of Pathology of the Humboldt University Berlin, GDR 1040 Berlin, Schumannstr. 20-21