Respiration of starfish oocytes during meiosis, fertilization, and artificial activation

Experimental

RESPIRATION

Cell Research 83 (I 974) 200-206

OF STARFISH

FERTILIZATION,

OOCYTES

AND

ARTIFICIAL

MARGARET Hopkins Marine Station of Stanford

DURING

MEIOSIS,

ACTIVATION

S. HOUK’

Universit),,

Pacific Grooe, Cal$. 93950, USA

SUMMARY Rates of oxygen consumption were measured in oocytes of the starfish Patiria miniatu prior to and after the initiation of meiotic maturation in response to I-methyladenine. No significant change in the rate of respiration was noted until after the completion of meiosis, at which point a two-fold increase in the rate of respiration was observed. The rate of oxygen consumption was also measured in response to fertilization and artificial activation with pronase. A transitory “burst” of oxygen consumption was noted in response to both stimuli. This “burst” is larger and of briefer duration in pronase-treated eggs. Possible interpretations of these phenomena are discussed

Sea urchin eggs experience an increased rate of respiration [8, 10, 17, 251 and of protein synthesis [7, 9, I I, 14, 231 after fertilization. These increases do not occur simultaneously; the respiratory increase precedes the increased rate of protein synthesis by several minutes [7]. The biochemical events immediately responsible for these two reactions also appear to be unrelated [5, 6, 12, 241. Yet, because they occur in response to the same stimulus, the increases in the rate of respiration and of protein synthesis have been interpreted as showing that the unfertilized egg is released from a state of general metabolic and synthetic inhibition by the penetration of the sperm [ 161. Unfertilized starfish eggs do not appear to share this release from inhibition. Neither the rate of respiration [IO, 191nor of protein synthesis [I 5, 211 changes in response to fertilization. Unlike sea urchin eggs, which mature in the ovary before spawning, starfish 1 Present address: Thimann Laboratories, University of California, Santa Crur, Calif. 95064, USA. Exptl Cell Res 83 (1974)

eggs mature after spawning induced by the hormone, I -methyladenine ( I -MA). Following spawning, maturation is completed whether or not the eggs are fertilized [ 13, 201. Therefore, although starfish oocytes are not immediately affected by fertilization, other events such as hormonal stimulation or the completion of meiosis could serve to release the oocytes from an inhibited state. Evidence, in fact, indicates that prior to treatment with l-MA starfish oocytes are in at least a partially inhibited state. In oocytes of Astcrias forhesii [4] and of Patiria miniata [21] protein synthesis increases at the time of germinal vesicle breakdown. Possibly, then, the eggs of Patiria and of other starfish are in an inhibited state in the ovary and experience a release from inhibition in response to hormonal stimulation rather than to fertilization. Previous studies of respiration in maturing starfish oocytes are contradictory. Borei [2] reports that the rate of respiration of mature eggs of Asterias glacialis is higher than that

Respiration of starfish oocytes of the primary oocytes. Using eggs of Asteriasforbesii other authors did not observe any increase [I, lo]. More recently, Schulz & Lambert

[19] have reported higher rates in maturing oocytes of Pisaster mhraceus and Patiria miniata but did not satisfactorily relate the increase to simultaneous meiotic events. Since the rate of respiration has long been a major criterion for deciding whether an egg inhibited. the present study is metabolically

of respiration

was designed to examine the respiratory rate of Patiria oocytes during I -MA-induced maturation, especially during those events which in other studies have been implicated in a release from inhibition. Fertilization, hormonal stimulation, the time of germinal vesicle breakdown, and the completion of maturation were specifically examined. An oxygen electrode was used to provide a continuous monitoring of respiratory rates so that any change might be correlated with concurrent cytological conditions in the oocytes. The results indicate that respiration is not affected, either by I-MA-induced meiosis or by fertilization. The respiration rate remains constant throughout meiosis, but rises 1.5 to two times after the eggs are mature. After fertilization there is a transitory “burst” of oxygen consumption similar to that reported by Horwitz [lo], but the rate immediately falls to the prefertilization level. The effects of artificial activation by pronase were also investigated and found to involve a respiratory “burst” of shorter duration and more accelerated rate than that of fertilization. MATERIALS

AND METHODS

Oocytes for the experiments described below were obtained by dissecting ovaries from futiria miniatu females collected at Pacific Grove. Calif. or at La Jolla, Calif. Experiments were conducted in September 1971 and May 1972. Results were consistent between the two periods of investigation.

201

Isolated oocytes were used in experiments involving respiration of primary oocytes and I-MA-stimulated oocytes. Oocytes were isolated by tearing the ovary with forceps and razor blade and gently freeing the cells by forcing them through a pipette. The oocytes were then washed and kept in 0.01 M Tris-buffered (I M Trisma base in sea water, I :99, adjusted to pH 8.0) Millipore-filtered sea water at 16’C. The oocytes were used within 1 h of isolation. For experiments involving fertilization the ovaries were minced and induced to spawn by treatment with several ml of I 10e5M I-MA. The maturing oocytes were then decanted into another container, washed, and used at the appropriate meiotic stage. Rates of oxygen consumption were measured with a Beckman oxygen analyzer and a platinum electrode in a water-jacketed cuvette maintained at 16°C. Output of the analyzer was recorded on a Sargeant SRL potentiometer recorder. The electrode was fitted with a rubber stopper which provided a tight seal inside the rim of the cuvette. I -MA, sperm, respiratory inhibitors, and pronase were introduced into the chamber through a slit in the stopper just large enough to admit the tip of a Pasteur pipette. Oocytes to be tested were allowed to equilibrate in the chamber in approx. IO ml of Tris-buffered, air-saturated, Millipore-filtered sea water before measurements began. Throughout the experiments the oocytes were stirred constantly by a magnetic stirrer except that stirring was stopped for IO-15 set after sperm were added to allow the eggs to fertilize. The extent of damage and I he state of maturation of the oocytes were observed during and after each experiment. After each experirnent the volume of the chamber contents was measured. The oocytes were then transferred to a hematocrit tube and centrifuged for 5 min at a uniform speed to determine the packed cell volume. To determine absolute rates of oxygen consumption, the Sargeant recorder was calibrated so that air-saturated Millipore-filtered sea water registered as 100 f’. and oxygen-exhausted sea water as O(‘,,. The oxygen cont. in air-saturated sea water was assumed to be 2.5 j 10e7 M/ml. The percentage of available oxygen which was consumed per unit time was calculated using a tangent to the recorder tracing at the point of interest. The absolute rate was calculated from the following formula: 0o total 0, consumed 2.5 M dissolved 0,jmI chamber vol in ml timein SG - vol of packed oocytes in ml

RESULTS Patiria oocytes consume oxygen at a rate of approx. l-5 x IO-IO moles/set/ml packed oocytes. This is somewhat lower than the rate reported by Horwitz for Asterius oocytes. Respiration was inhibited 50 Y, by I x lO-6 M NaCN and 700:, by I x 10m6M antimycin A. Exptl Cell Res 83 (1974)

202

Margaret S. Houk

Table 1. Oxygen consumption in I-MA-stimulated

and unstimulated oocytes

Rate of 0, consumed

Ratio to initiala rate

(M O,/sec/ml packed oocytes) Sample

Unstim

Unstimulated oocj,teJ Expt 1 2 3 4

IO-‘”

0 min

10 min

20 min

30 min

1.85 2.02 I .20 1.78

1.60 1.95 1.25 1.76

1.60 2.00 1.27 1.75

1.60 2.10 I .30

0 mini Initial

Average 95’ confidence limit L l-MA-stimulated oocytes Expt I 1.85 2 1.96 3 2.02 4 1.78

1.85 1.98 I .82 1.72

1.85 2.06 1.98 I .72

1.85 2.06 2.00 1.60

1.72 2.06

1.oo 1.01 0.90 0.97 Average 0.97 95’ confidence limit + 0.06

IO min/ Initial

20 min/ Initial

30 min/ Initial

0.87 0.97 1.04 0.99 0.97 0.09

0.87 0.99 I .06 0.98 0.98 0.11

0.87 I .04 I .08

I .oo 1.05 0.97 0.97 1.oo 0.05

I .oo I .05 0.99 0.90 0.98 0.09

0.93 1.05

a The initial rate for unstimulated oocytes refers to the first reading taken. In l-MA-stimulated refers to the rate prior to addition of I-MA.

The latter inhibitor was added to the chamber from a 1 x 10~~ M stock solution in ethanol. At these concentrations ethanol did not affect the respiratory rate. The rate of oxygen consumption in untreated oocytes is constant for the first 30 min of measurement following equilibration in the chamber (table 1). Single experiments of much longer duration were not attempted since the oocytes were often damaged by long periods in the chamber. Primary oocytes induced to complete meiosis with the chamber by adding 0.2 ml of 1 x 10~~ M l-MA showed no significant change in the rate of oxygen consumption during the first 30 min after addition of I-MA (table 1). Since this includes the period during which the germinal vesicle breaks down and protein synthesis begins to increase in stimulated oocytes, there is no rise in the rate of respiration associated with these processes. To determine whether oxygen consumption undergoes any changes during meiosis, approx. 5 ml oocytes were isolated and kept as a Exptl Cell Res 83 (1974)

1.oo 0.22

0.99 0.51 oocytes it

stock on a water bath at 16°C. One-fifth of the oocytes were withdrawn and placed in the electrode chamber. After several readings were taken, l-MA was added simultaneously to the chamber and the stock suspension of oocytes. 4.5-60 min later, the first aliquot was removed from the chamber, and another was added from the stock. A replacement of the oocytes in the chamber was made every 45-60 min throughout the experiment. Readings were taken only on the oocytes which were at least 90% healthy and maturing normally. The absolute rate of oxygen consumption for 4 expts and a comparison of each to the rate of oxygen consumption of unstimulated oocytes in the respective experiments are presented in table 2. There is no significant change in the rate of respiration until 170 min after addition of l-MA. At this time there is a rise in the rate of respiration by 50-100 y. of the rate in primary oocytes. At 170 min the oocytes had already completed meiosis and were mature eggs. A t test indicates that

Respiration

Table 2. Oxygen consumption

during meiotic

of starfish oocytes

203

maturation

Rate of 0, consumption in M/set/ml packed oocytes .<10-l” Sample Time Phase Expt I 2 3 4

0 min Pro. 1

20 min Pro. 1

60 min Met. 1

100 min Pro. 2

120 min Met. 2

I70 min Mature

200 rnin Mature

1.72

1.60

2.45

1.92 5.3 4.6

1.55 5.8 4.9

1.22

2.25 1.57 a a

1.92 1.87 5.7 7.6

2.48 2.92 8.1 5.8

= 4.00 a 7.1

5.6 a

Ratio of rate at indicated time to rate at 0 time 20 min Expt I 2 3 4

Average 95” limits of confidence

0.93 0.81 1.09 1.07 0.98

0.19

60 min 1.42

100 min 1.31

120 min 1.11

170 min 1.44

200 min a

0.64 1.05 a 1.04

0.82 a. a

0.95

1.52

2.31

1.08

1.53

a

1.65

1.26

1.54

1.06

1.20

1.44

1.92

0.59

a

0.43

0.17

a

a Confidence limits were not determined when only two samples were available. Data are missing because readings were not used if more than 10 “,, of the oocytes were damaged.

the 170 min data differ significantly from the values for primary oocytes at a confidence interval of 0.85. Results of experiments in which the eggs were fertilized are shown in table 3. After oocytes or eggs had been allowed to equilibrate in the chamber, several drops of sperm suspension were introduced through the slit in the stopper. The stirrer was stopped to allow sperm-egg contact and started again IO-15 set later. The rate of oxygen consumption remained constant for 60 set, then rose abruptly to a peak at 150 sec. The peak was 3 times as high as the rate in unfertilized oocytes and coincided in time with the first appearance of the fertilization membrane. At this time microscopic observations revealed a large amount of material being released into the perivitelline space, presumably from the breakdown of the cortical granules. The rate of oxygen consumption fell almost as fast as it rose, returning to the pre-fertilization level by 5 min after sperm addition. Oocytes were fertilized at different

times during meiosis with no significant differences in the respiratory response to fertilization. A parthenogenic activator of starfish eggs, pronase, was tested for its effects on the rate of oxygen consumption. Pronase (Calbiothem.) was added to the chamber from a stock solution of 10 mg/ml. The cont. in the chamber approximated 100 pug/ml. Pronaseactivated oocytes exhibit a respiratory “burst” which is higher, quicker, and of briefer duration than the comparable response to fertilization (fig. I). It begins within 30 set of treatment, reaches a peak at 60 set and return treatment, reaches a peak at 60 set and returns to the base rate by 120 set after addition of pronase. The fertilization membrane also elevates faster in pronase-treated oocytes, beginning 45 set after treatment as opposed to 90 set in fertilized oocytes. For both types of activation the initiation and duration of the respiratory peak correspond to the elevation of the fertilization membrane. The area under the curve was estimated for Exptl Cell Res 83 (1974)

204

Margaret S. Houk

Table 3. effect of fertilization

on the rate of ox)‘gen consumption

Rate of oxygen consumption (M O,/sec/ml packed oocytes) j 10-l” Unfert.

Fert. I min

Primary oocytes 2.43 2.43 3.21 3.27 3.34 3.12

Ratio to the initial

rate

Fert. 2 min

Fert. IO min

I min/ Unfert.

2 min/ Unfert.

IO mini Unfert.

8.10 12.30 Il.25

2.52 2.90 2.92 Average

1.00 I .oo I.11 I .04

3.33 3.76 3.50 3.50

I .04 0.89 0.89 0.94

9.10 7.80 8.60

5.09 3.45 4.05 Average

I .oo 1.34

5.70 2.56 I .95 3.40

2.95 I.13 0.92 1.61

Secondary oocytes 1.72 1.I2 3.04 4.20 4.40

1.17

3 expts in which the eggs were fertilized and 3 in which they were activated with pronase. The rates for each expt were plotted on graph paper of uniform thickness using the same scale, and the part of the curve judged to constitute the “burst” of oxygen consumption was cut out and weighed. The average wt for fertilized eggs was 0.0925 g and that of the pronase-treated eggs was 0.093 1 g. Therefore, although the shape of the curve of oxygen consumption differs for the two types of treatment, the total amount of oxygen consumed during the “burst” does not differ significantly between fertilized and pronaseactivated oocytes.

eggs are not released from inhibition by the sperm in the way that sea urchin eggs are. Changes in the rate of respiration u’uring meiosis Since an increase in the rate synthesis has been reported at germinal vesicle breakdown in Asterias forbesii [4] and Patiria

36

of protein the time of oocytes of miniata [21]

!

% A

DISCUSSION Rate of respiration following fertilization The rate of oxygen consumption is not permanently changed by fertilization of either primary or secondary oocytes of several species of Aster& [2, 10, 221 and Pisaster ochraceus and Patiriu miniata [19]. My data confirm these observation for P. miniata. Since fertilization also has no effect on the rate of protein synthesis [15, 211, starfish Exptl Cell ReA 83 (1974)

Fig. 1. Abscisa: time from fertilization (A curves) and from pronase activation (B curves) (min); ordinate: M 0, consumed/set/ml packed oocytes ., ,o-10, Oxygen consumption following fertilization and pronase activation in oocytes of Patiria miniafa. Two experiments are shown for each stimulus. A, time at which sperm were added; B, time of addition of pronase.

Respiration of starfish oocytes starfish oocytes might be stimulated by the hormone in a manner similar to the postfertilization response in sea urchins. The increased rate of protein synthesis is not, however, accompanied by increased respiration and is therefore an isolated response to the hormone and not part of a general process of activation. An increased rate of respiration has been reported after completion of meiosis in eggs of Asterias glacialis [2] and Asterias forbesii [3]. In the latter species no rise was found by Boell et al. [I] or Horwitz [IO]. Recently, Schulz & Lambert [19] have reported a rise in the rate of respiration in oocytes of Pisaster ochracel,.~ and Patiria miniata with 1 h of treatment with I-MA. Schulz & Lambert suggest that the respiratory increase is concurrent with germinal vesicle breakdown and may be a result of germinal vesicle breakdown. My results indicate that if increases occur in the rate of respiration during maturation of oocytes of Patiriu miniata, they happen long after germinal vesicle breakdown is complete and are more closely associated with the completion of meiosis. My results support the possibility that an increased rate of respiration follows the completion of meiosis, but the increase is small and could have been overlooked by some of the previous investigators. As no increase in the rate of protein synthesis occurs at this time [21], such a stimulation of respiration is again an isolated response rather than a part of a general release from inhibition as occurs in sea urchin eggs at fertilization. Mechanisms which mediate the postfertilization activation of respiration are well studied in sea urchin eggs. The increases apparently occur through higher availability of substrate and enzymes [12] and of coenzymes [5, 61. It would be informative to establish the nature of the controls over similar events in starfish oocytes. This inform-

205

ation would help in determining the degree of similarity of biochemical controls over early development of various deuterostomes. Nature of post-fertilization oxygen consumption Both sea urchin eggs and starfish oocytes exhibit a post-fertilization “burst” of oxygen consumption beginning with the first appearance of the fertilization membrane and continuing while the membranes are rising [8. IO, 171. In pronase-treated oocytes of P. miuiata the membranes appear more quickly and rise more quickly than they do in fertilized oocytes. The “burst” also begins more quickly and is more abrupt in pronase-treated oocytes. The same total amount of oxygen appears to be consumed as in fertilized oocytes. The timing of the “burst” suggests that it is related in some way to the cortical reaction. In sea urchin eggs, the elevation of the fertilization membrane is inhibited by respiratory uncouplers and is therefore thought to be an energy-requiring process. The actual energyrequiring step appears to be propagation of the fertilization “wave”, which passes over the egg surface in advance of the lifting of the membrane [18]. Since this “wave” occurs very near the time of the “burst” in oxygen consumption, the two processes may be related. Alternatively, the “burst” may result from release of cortical granule products into the perivitelline space during membrane formation. Some of these products may be oxidized extracellularly, with a consequent “burst” of non-metabolic oxygen consumption. The timing, suddenness, and short duration of the “burst” support such a theory, as does the occurrence of an even more abrupt “burst” in pronase-treated oocytes which utilizes the same absolute quantity of oxygen as the post-fertilization event. An understanding of the nature of the Exptl Cell Res 83 (1974)

206 Margaret S. Houk could be informative respiratory “burst” about the mechanism of fertilization and of subsequent blocks to polyspermy. If the oxygen consumption is extracellular, the substrate could be instrumental in hardening of the fertilization membrane. If it is metabolic, the activity which requires such a sudden expenditure of so much energy must represent an important part of the process of fertilization. I thank Dr David Epel for his help and advice concerning the work reported in this paper and for his criticism of the manuscript. This work was supported in part by NSF grants GB-8002 and GB-16155.

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Exptl Cell Res 83 (1974)

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processes (ed M Locke) p. 170. Academic Press. hew York, (1967). 25. Warburg, 0, 2 physiol Chem 57 (1908) I Received June 7, 1973