Effects of senescence on somatic cell physiology in the green alga Volvox carteri

Copyright @ 1982 by Academic Press. Inc. All rights of reproduction in any form reserved 0014-4827/X2/070039-0762.00/O

Experimental Cell Research 140 (1982) 39-45

EFFECTS

OF SENESCENCE IN THE GREEN

ON SOMATIC ALGA

J. POMMERVILLEL

VOLVOX

CELL

PHYSIOLOGY

CARTERZ

* and G. KOCHERT

Department of Botany, University of Georgia, Athens, GA 30602, USA

SUMMARY In every generation resulting from asexual reproduction, the somatic cells of the green alga Volvox carteri undergo synchronous senescence and cell death. Although the somatic cells of the asexual and sexual (female) generations of V. carteri f. weismnnniu ceased growth 72-96 h after the cells had stopped dividing, the somatic cells in the sexual spheroid remained alive approx. 96 h longer than comparable cells in the asexual spheroid. Measurement of several environmental factors demonstrated that senescence was not caused by extrinsic factors, such as changes in the light period, temperature, or culture medium. If these factors (light, temperature) were purposely altered, the timing of viability decline changed. Other changes occurring with senescence included a decreased rate of [W]bicarbonate assimilation after 96 h and a rapid decline in total soluble cell protein. The addition of the protein synthesis inhibitor, cycloheximide, prolonged the onset of senescence and delayed the decline in somatic cell viability. By adding cycloheximide at different times during senescence, we were able to show that the drug had less effect after % h than it did when added at 72 h. These results rule out extrinsic events governing senescence and are consistent with our idea that senescence in V. carteri is due to an intrinsic, sequential genetic program.

The loss of inherent cell functions and replicative capacity (senescence) in most animals and many plants can be the result of gradual changes within various cells and tissues [3, 93. In plants, these deteriorative effects appear to be the result of internally regulated senescence events [9]. Leaf senescence has been studied in detail [see 17, 181, especially with regard to cellular and biochemical changes. It has been stated that leaf senescence is the result of genetic control [2, 17-201 and is an expression of the interaction between the nuclear genome and the genome of the chloroplast [19, 201. But leaf senescence, as well as whole organism senescence, can be difficult to analyze in terms of internal, genetically programmed events. This is partly due to problems in studying the interactions and

relationships between different cell types and tissues, and the varying degrees of senescence between interacting tissues. It would be easier to study an organism where there are fewer cell types, where the life cycle is fairly short, and where senescence occurs in a predictable fashion from generation to generation while in culture. A very interesting organism to carry out physiological studies on senescence is the green alga Volvox carteri. This eukaryotic, phototrophic microorganism contains only two cell types--about 4000 somatic cells that make a hollow ball (spheroid) and 8-16 asexual reproductive structures (gonidia) that reside inside the spheroid. Each ’ Present address: Department of Biology, Texas A & M University, College Station, TX 77843, USA. ’ To whom offprint requests should be addressed. tip

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gonidium has the ability to form a new daughter spheroid by differentiating into new gonidia and somatic cells that are identical to the parent [7, 83. After the required number of somatic cells have been produced by cell division, they stop dividing, increase rapidly in size, and eventually undergo senescence and cell death [S]. In every asexual generation, all the somatic cells die. The survivorship curves for the somatic cells from the asexual spheroid in V. carteri f. weismannia [12] are exactly the type that one would expect for an organism undergoing programmed senescence and aging [3, 6, 91. Morphological changes that accompany senescence in V. carteri f. weismannia demonstrate that senescence begins within 72-96 h after the somatic cells stop dividing and that the viability decline of the somatic cells begins about 144 h after the cessation of division. The death of the somatic cells is not due to the build-up of toxic materials or a lack of nutrients in the culture medium [ 121. We have suggested [5, 121 that senescence is an internal, programmed event(s). Therefore the present study was undertaken to discover if there are environmental conditions that are responsible for or influence senescence of the somatic cell, and to see if physiological changes accompany those morphological alterations described previously [12] for the somatic cells of V. carteri f. weismannia. A preliminary account of part of this work has been presented [ 131. MATERIALS

AND METHODS

Growth conditions The MO-4 strain of Volvox carteri f. weismannia was used for all the experiments reported here. Stock cultures and the establishment of culture synchrony have been previously described [8, 121.Cell size was Exp Cell Res 140 (1982)

determined with an optical micrometer attached to the eyepiece of a Zeiss research microscope.

Environmental parameters Spheroids from synchronously growing cultures were inoculated into fresh Volvox medium [14] and kept in constant light, unless otherwise mentioned. The effect of several environmental parameters on senescence and somatic cell viability were examined. These included: (1) the insertion of a 24-h dark period by wrapping the cultures in foil; (2) altering the normal growth temperature of 27°C by placing the cultures in temperature-regulated, water-jacketed spinner flasks (Beico) in the fight; or (3) growth of the culture at a pH different from that of the normal culture medium (pH 8). At 24-h intervals after inoculation of the spheroids into the culture medium, aliquots were removed aseptically and the viability of the somatic cell population determined by trypan blue exclusion [ 121.

Photosynthetic rate Cells were released from the sheath using a Branson Sonifer Cell Disruntor. This freed the cells from the sheath without significant loss of cell numbers or cell death. as determined bv trvnan blue exclusion. Somatic’cells at a concenLat{ort of 3X 105 were inoculated into 5 ml of Volvox medium containing 0.5 fi.Ci [W]bicarbonate/ml under conditions of continuous light and bubbling. At 15, 30, 45, and 60 min after i&ulation, sampies were removed, filtered onto 0.45 pm Metricel filters (Gelman Sci., Inc.), and washed with Volvox culture medium. The filters were treated with 0.5 N HCI for 2-3 h. dried, and counted in the presence of a non-aqueous scintillation cocktail in a Packard Tri-Carb liauid scintillation snectrometer. Controls consisted of’(l) vials without cells; (2) vials without PClbicarbonate; and (3) vials containing killed somatic cells.

Cell protein Total cellular protein in the somatic cells was determined by the method of Bradford [ 11.

Cycloheximide experiments The effects of cycloheximide (CHX) on somatic cell development and senescence were examined by inoculating whole spheroids into Volvox culture medium containing 0.1 pg/ml CHX under conditions of constant light. At 24-h intervals, aliquots were removed and their viability determined by trypan blue exclusion. At specific times, the entire culture was washed aseptically with Volvox medium by filtration through Miracloth (Chicopee Mills) to remove CHX. The spheroids were resuspended in Volvox medium without CHX for further monitoring of somatic cell development and viability. All results represent the means determined from at least three separate experiments.

Senescence in Volvox

41

Fig. I. Survival curves for the somatic cells from x, asexual spheroids; 0, female spheroids of V. carteri f. weismannia, and 0, somatic cells from asexual spheroids of V. carteri f. nagariensis.

Fig.

RESULTS Time course of the senescence process We have previously shown [12] that senescence in somatic cells of asexual spheroids of V. carteri f. weismannia begins about 72 h after the cessation of division, whereas cell viability begins to decline at about 144 h. The gonidia in asexual spheroids can be induced by a pheromone to become sexual spheroids containing eggs [8]. When the somatic cells of the female sexual spheroid of V. carteri f. weismannia were analyzed, we found that these cells lived much longer than comparable somatic cells from asexual spheroids grown under identical conditions (fig. 1). The overall shape of the survivorship curve is very similar, however. This agrees with microscopic observations of cultures containing female and asexual spheroids. In such cases one notes that female spheroids persist for more than one asexual generation. In addition, somatic cells from asexual spheroids from another form of V. carteri (f. nagariensis) died much sooner than comparable V. carteri f. weismannia cells. Measurement of cell size and surface-volume ratios of the somatic cells from asexual and sexual spheroids of V. carteri f. weismannia showed that somatic cell growth ceased be-

tween 72-96 h after the somatic cells stopped dividing (table 1). After this time, the whole spheroid continues to increase in size, however, due to expansion or addition of sheath material between the cells. Several imposed perturbations to the normal culture conditions were shown to change the time of viability decline. If spheroids containing 72 h somatic cells were placed in the dark for 24 h, the decline in viability did not begin until about 168 h, 24 h later than if the cells were in continuous light (fig. 2). Gonidial development into new daughter spheroids also was delayed 24 h. If the dark period was given at times later

2. Effect of a 24 h dark period on viability decline in V. carrier f. weismannia. x , Cells always in the light; somatic cells in the dark from 0, 7296 h; .,96-120 h; A, 120-144 h.

Table 1. Changes in somatic cell diameter and cell-cell distance during development and senescence in V. carteri f. weismannia Cell-cell distance km) Cell diam. Cf.4

Anterior

Posterior

24 48

3.3 4.4


Cl 1

z 120 144 168

2:: 2.:

it:?0 8” 9.9?0:7 20.0f0.9 24.0f0.8

Time (hours)

5:5

1

:::*o 5 5.9*0:7 11.2kO.5 13.2f0.6

a SE. EXP Cell Res 140 (1982)

42

Pommerville and Kochert

Fig. 3. Effect of temperature on viability in somatic cells of V. carteri f. weismannia. Normal growth temperature of X, 27; 0, 35; l ,40; A, 20°C.

Fig. 5. Assimilation of [Tlbicarbonate into somatic cells of V. carteri f. weismannia during development

and senescence. 0,24; 0, 48 h; X, 72; V, 96; A, 120; A, 144 h somatic cells.

than 72 h, less or no effect of the dark fluctuation. However, if the Volvox medium was prepared at different pH values, period occurred (fig. 2). Alterations in temperature influenced the viability decline was altered. At a more decline in viability of the somatic cells. acidic pH, the gonidia developed slower Normally the cultures are maintained at and the decline in somatic cell viability 27°C. If the cultures were grown at tem- occurred later than in control cultures at peratures above 27”C, the somatic cells died pH 8 (fig. 4). When the pH of the medium sooner and the gonidia developed faster was more alkaline, the cells died slightly (fig. 3). At lower temperatures than 27”C, sooner. the somatic cells died later and the gonidia Photosynthetic rate developed slower. One environmental parameter that could All the above results point to internal confluctuate is the pH of the culture medium trols on senescence and death of the soprior to, during, or resulting from senes- matic cells in V. carteri f. weismanniu. cence. Monitoring the pH of the culture One internal alteration resulting from senesmedium during growth and senescence of cence could be the loss of photosynthetic the somatic cells showed no significant activities [12]. Thus the ability of the so-

Fig. 4. Survivorship curve for somatic cells from V. carteri f. weismanniu grown in culture at differing pH values. x , Normal pH 8; 0, pH 6; 0, pH 7; A, pH 9. Exp Cell Res 140 (1982)

Fig. 6. Change in [‘T]bicarbonate

assimilation for 60 min with different ages of somatic cells of V. carteri f. weismannia.

Senescence in Volvox

Fig. 7. Changes in total soluble protein during development and senescence of somatic cells in V. carteri f. weismannia.

matic cells to assimilate [14C]bicarbonate over a short pulse period of 15-60 min was used as a measure of the photosynthetic rate. These experiments demonstrated that the cells rapidly took up the bicarbonate early in development but this ability quickly declined in older cells (figs 5, 6). Cycloheximide studies Measurement of total soluble cell protein in the somatic cells of V. carteri f. weismannia during growth and senescence showed a sharp decline after 120 h (fig. 7). This rapid decline in the level of total cellular protein led us to examine the sensitivity of somatic cell development to the protein synthesis inhibitor cycloheximide (CHX). At a CHX concentration of 0.1 pg/ml, viability decline was delayed for several days (fig. 8), and gonidial development was inhibited as well. CHX treatment of spheroids did not kill the cells since they remained viable according to trypan blue exclusion measurements. At specific periods after incubation in CHX, the spheroids were removed from the drug, washed in Volvox culture medium, and resuspended in culture medium without the inhibitor. These spheroids then resumed gonidial de-

43

Fig. 8. Effect of cycloheximide (CHX) on somatic cell viability in V. carteri f. weismannia. At 72 h (arrow), CHX was added and the viability of the somatic cells determined at 24-h intervals. X, No CHX added; 0, 0.1 pg/ml CHX; 0, cultures where the CHX was removed at 144 h (arrowhead) and viability determined by dye exclusion.

velopment and differentiation, and the somatic cells began to show a decline in viability sooner than those cells still in CHX but later than those cells without the drug (fig. 8). By initially adding CHX at later and later times, it was determined that beyond 96 h, CHX had less effect in prolonging viability (fig. 9). Measurement of total chlorophyll [see 121after washing the spheroids out of CHX showed that degradation of chlorophyll was inhibited by CHX treatment (results not shown). Similar experiments were done with the mitochondrial and chloroplast protein synthesis inhibitor chloramphenicol (CAP). Addition of CAP prevented gonidial de-

Cell ape ,h)

Fig. 9. Effect of time of addition of cycloheximide (CHX) to cultures of V. carteri f. weismannia. CHX added at: 0,72; A, 96; A, 120 h; x, no CHX added. Exp Cell Res 140(1982~

44

Pommerville

and Kochert

velopment and differentiation, but did not ration of chloroplast structure in the soextend or reduce significantly the senes- matic cells. Measurement of total somatic cell chlorophyll showed a decline during cence period of the somatic cells. this period [12] which correlates with the decline in [‘4C]bicarbonate assimilation reDISCUSSION ported in this paper. Environmental factors are constant in our Survivorship curves of all V. carteri varieties are similar in having a rapid decline in standard culture conditions for measuring somatic cell viability. Since they all show senescence of the somatic cells. The survival rate of these cells can be shortened declines at different times, environmental factors appear not to control senescence. or lengthened by manipulating the standard These curves seem to be what one would conditions. Light shows an inhibitory reexpect for an organism undergoing pro- sponse toward viability decline if the dark grammed senescence and aging [3, 6, 91. period is given at 72 h. This probably reUnder our standard conditions for growth sults from the slowing of development due of synchronous cultures, the entire process to reduced photosynthesis since the whole of somatic cell development and differentialife cycle is lengthened. However, since this tion, from the cessation of somatic cell light effect is only seen at 72 h and not division to death of the somatic cell popu- later, the period between 72 h and 96 h is lation in asexual spheroids of V. carteri f. again seen as a critical time for the onset weismannia, takes about 192 h. Viability of senescence and the viability changes that decline occurs only over the period from result from it. Also, when spheroids of V. 144 to 192 h, yet senescence, as measured carteri f. weismannia are grown under a by decreased levels of cellular protein and normal light-dark cycle to maintain synlowered photosynthetic rates, begins about chrony in stock cultures, the survivorship 72-96 h after the somatic cells stop divid- curve is identical to that in total light [12]. ing. This interval of 72 h between senes- It could be suggested that somatic cell death cence onset and the beginning of viability results from factors produced by the goloss may be an important time span to in- nidia, since they are inside the spheroid. vestigate what biochemical changes occur If the gonidia are removed from the spheas a result of senescence. roid, the rate of viability decline of the The failure of cells to maintain homeo- somatic cells does not change [4], and if stasis has been implicated as an important old spheroids are mixed with cultures confactor in senescence [16]. Cessation of taining young spheroids, all undergo senesgrowth as a result of some developmental cence at their normal rates. Therefore only event(s) reflects a change in cell metabolism internal events within the somatic cells and could lead to senescence. Measurement appear responsible for senescence in this of cell surface-volume ratios and cell size alga. indicate that cell growth stops around 72In terms of macromolecular synthesis, 96 h, which is the period when senescence polypeptide changes could occur within the can first be detected. Electron microscopy somatic cells that are responsible for seshowed changes in somatic cells during this nescence and the observed viability decline. time [12]. There was an increase in cyto- Measurement of total protein showed a plasmic lipid bodies, followed by a deterio- sharp decline correlating with the reported Exp Cd

Rcs 140 11982)

45

Senescence in Volvox decline in 35Sincorporation into protein in V. carteri f. nagariensis [5]. This, along with the electron microscopy results [12], shows that senescence, at least in part, is a degradative process. Research with other plant systems has provided evidence for an increased rate of proteolysis during senescence [IS], although a reduced rate of protein synthesis also could contribute to the process. Like some other higher plant systems that have been used for senescence studies [ 10, 111,CHX prolongs senescence, viability, and the loss of chlorophyll in V. carteri f. weismannia without irreversibly inhibiting gonidial development and differentiation. The CHX results strongly suggest that a protein (or proteins) is involved in senescence and cell degradation. Proteolytic enzymes could be one way of accounting for the protein degradation such that CHX blocks this synthesis. Alternatively CHX may block the synthesis of an enzyme involved in ‘activating’ a proteinase. Since all proteins are not degraded at the same rate; proteolysis must be controlled and not an uncontrolled degradative event, again suggesting genetic control. Before we can study the factors that cause senescence and aging in the somatic cells of V. carteri, and before we can understand the possible role of genetic elements, we must understand the morphological and physiological events and functions that decline or change during senescence. We believe our results further support, and are consistent with the idea that there is an intrinsic, sequential genetic program for somatic cell senescence in V. carteri.

Printed

in Sweden

This work was supported through NIH grant 5 RO 1 AG 00127-02to G. K.

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Cell

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140 (19821