- Email: [email protected]
Hormonal effects on RNA synthesis by stage 6 oocytes of Xenopus laevis
DEVELOPMENTAL
Hormonal
BIOLOGY
47, 384-393 (19%)
Effects on RNA Synthesis by Stage 6 Oocytes laevis l
MICHAEL J. LAMARCA*, MARJORIE C. STROBEL FIDLER, L. DENNIS Department
of Biological
KIRSTEN Sciences, Purdue
KEEM University
West Lafayette,
Indiana
of Xenopus
SMITH,
AND
47907
Accepted June 26,1975 RNA synthesis has been studied in “large” oocytes of Xenopus laevis, both as a function of time after injection of females with human chorionic gonadotropin (HCG) and in relation to the induction of maturation with progesterone in vitro. Rates of RNA synthesis were measured by analyzing the kinetics of incorporation of exogenous 13Hlguanosine, and microinjected [3H]- or [*%IGTP, into acid-precipitable material, coupled with measurements of precursor pool specific activity. The kinetics of incorporation into RNA of injected precursor are biphasic, indicating the synthesis of both stable and unstable RNA species. Estimates of the total rate of synthesis (stable and unstable) were derived from fitting a linear function to data over the first 60-90 min, while a linear function fit to the data beyond 90 min represented largely the synthesis of stable RNA species. Exposure of oocytes to progesterone had no effect on initial synthetic rates, but maturing oocytes synthesized stable RNA at 1.4-1.6 times the rate in control oocytes. A comparison of data obtained with oocytes from unstimulated (no prior HCG treatment) and HCG-stimulated females indicated that HCG has no substantial effect on rates of RNA synthesis. The signilicance of continued RNA synthesis in large full grown oocytes is discussed. INTRODUCTION
It now appears certain that amphibian oocytes remain synthetically active as they complete their growth phase, both in terms of protein and RNA synthesis (review by Smith, 1975). Studies on protein synthesis, primarily with Rana pipiens oocytes, further demonstrated that the level of synthetic activity is under hormonal control, especially during the period of oocyte maturation (see Smith, 1975). Similar studies on RNA synthesis, almost exclusively with Xenopus laevis oocytes, have been incomplete and somewhat contradictory. Brown and Littna (1966) reported that during the period of ovulation, Xenopus Zaevis oocytes synthesized nanogram quani Research supported by grants from the USPHS (No. HD04229) and the National Science Foundation (No. GB39971). This work was initiated while the principal investigator was an NSF. Science Faculty Fellow in the Department of Biological Sciences, Purdue University, West Lafayette, Ind. 47907. 2 Present address: Department of Biology, Lawrence University, Appleton, Wis. 64911. 384 Copyright 0 1975 by Academic Ress, Inc. All rights of reproduction in any form reserved.
tities of DNA-like RNA, presumably in response to the gonadotropin (HCG) used to induce ovulation. Subsequently, Mairy and Denis (1971) reported that large oocytes (1.0-1.2 mm diameter) taken from females recently induced to ovulate with HCG incorporated greater amounts of exogenous guanosine into RNA than similarsized oocytes taken from females not treated with HCG. However, potential permeability changes in response to the hormone were not monitored, and increased incorporation could have resulted from greater uptake of precursor in oocytes from HCG-treated females (see Mairy and Denis, 1971). Recently, both uptake and incorporation of radioactive precursors into RNA have been measured in ovarian follicles removed from HCG-stimulated females (Colman, 1974) and in ovarian tissue exposed to HCG or progesterone in vitro (Wassarman and Masui, 1974). Neither parameter was obviously altered during long-term (24 hr) or shorter (6 hr) pulses to radioactive precursors, but the differential behavior of oocyte versus so-
LAMARCA et al.
Hormonal
matic follicular components was inadequately quantitated. Recently, we studied RNA synthesis in the largest oocytes taken from Xenopus females that had not been induced to ovulate (LaMarca et al., 1973). Rates of RNA synthesis, determined from kinetics of guanosine (GTP) incorporation into RNA and changes in precursor pool specific activity, were at least as great in these large oocytes as in smaller oocytes (500-600 pm diameter). We now report similar experiments in which rates of RNA synthesis were measured in isolated “large” oocytes, both as a function of time after injection of females with HCG and in relation to the induction of maturation with progesterone in vitro. MATERIALS
AND
METHODS
a. Animals Mature Xenopus laevis females were obtained directly from South Africa and maintained as previously described (Webb et al., 1975). Animals classified as unstimulated refer to females collected directly from the wild and used after maintenance in the laboratory for varying time periods (minimally 4-6 weeks). They had not been induced to ovulate, and their ovaries contained white banded oocytes (see Dumont, 1972). Stimulated females had been induced to ovulate previously by injection of HCG (Antuitrin, Parke-Davis) into the dorsal lymph sac; 500 IU of HCG followed by an additional injection of 250 IU the following day. b. Preparation
of Oocytes
Ovarian lobes were removed from pithed females and were washed extensively in antibiotic Ringer’s solution as described (LaMarca et al., 1973). Oocytes were manually dissected from their follicles and adhering follicle cells removed by Pronase digestion (LaMarcaet al., 1973). All experiments were conducted on oocytes with diameters about 1.2 mm or greater. With
Effects
on Oocyte RNA
Synthesis
385
unstimulated females, the oocytes always contained white equatorial bands. After ovulation, white banded oocytes were no longer present and the remaining population of large oocytes, still about 1.2 mm in diameter, correspond more nearly to stage 5, as described by Dumont (1972). However, both these oocytes and the white banded ones respond similarly to progesterone (Reynhout et al., 1975). Maturation was induced by exposure to progesterone (5 pglml) for 10 min. Greater than 99% underwent GVBD, judged externally by the appearance of a circular whitish area in the animal hemisphere (Reynhout et al., 1975) and verified by dissection of selected oocytes. All operations were performed at a temperature of approximately 20°C. c. Uptake and Incorporation tive Precursor
of Radioac-
Follicle-free oocytes were incubated in sterile antibiotic Ringer’s solution containing 100 &i/ml of L3H]guanosine (NEN, tetrasodium salt, S3H, 6.5 Ci/mmole). At hourly intervals, groups of 10 oocytes were removed, rinsed, and either homogenized or soaked overnight in 0.5 N perchloric acid (PCA). Results were comparable with either procedure. Acid-soluble radioactivity represented total uptake. Radioactivity incorporated into RNA at each time point equaled the fraction of acid-insoluble radioactivity hydrolyzable by 0.3 N KOH as described (LaMarca et al., 1973). This procedure could overestimate rates of synthesis, since unincorporated nucleoside phosphates trapped in the acid-precipitable pellet would also be released by alkaline hydrolysis (see Emerson and Humphreys, 1970). However, results from these experiments are not significantly different from those obtained under conditions in which this problem has been taken into account. In most experiments, oocytes were injected directly with 15-20 nliters of 13HlGTP (NEN, tetrasodium salt, 5.28 Ci/mmole) or [%]GTP (Amersham-
386
DEVELOPMENTAL BIOLOGY
Searle, 500-545 mCi/mmole). At appropriate times after injection, oocytes were either homogenized in 0.5 iV PCA, followed by collection of precipitates on Millipore filters (HAWP, 45 pm pore) or were placed into cold 0.5 N PCA for overnight extraction of unincorporated material. Results with either procedure were essentially identical. In most experiments, filters or intact oocytes were combusted, without prior alkaline hydrolysis, and radioactivity incorporated into RNA at each time point was taken to be acid-precipitable radioactivity remaining after subtraction of acid-precipitable radioactivity present at zero time after microinjection (LaMarca et al., 1973). In selected experiments, injected oocytes were further hydrolyzed in alkali, and radioactivity in 2’,3’-GMP was determined. To avoid exchange of tritium with water during alkaline hydrolysis, oocytes were injected with [14ClGTP. At various times after injection, groups of 15 oocytes were placed into cold (4°C) 0.5 N PCA, extracted overnight and then divided into two groups. One group of five oocytes was combusted directly and counted. The remaining 10 oocytes (at each time point) were hydrolyzed in 0.3 N KOH for 18 hr at 37°C. The KOH-soluble material was collected, neutralized with sulfuric acid and, after addition of OD quantities of 2’- and 3’GMP and 5’-GTP, the sample was lyophilized. Individual samples were resuspended in water, and aliquots were spotted directly on Whatman 3MM paper. Nucleotides were separated by descending chromatography in a high salt solvent (solvent D, Bennett and Gilham, 1975)consisting of water (100 ml) and (NH,),SO, (40 g) adjusted to pH 8.5 with concentrated NH,OH. Relative to 5’-GTP, 2’- and 3’GMP migrated with R, values of 0.83 and 0.68, respectively. However, 5’-GMP also cochromatographed with 2’-GMP. Hence, to determine the fraction of radioactivity present only in 2’,3’-mononucleotides from RNA, the 3’-GMP spot (identified by
VOLUME 47, 1975
marker 3’-GMP) was cut out and counted directly in toluene-PPO. Assuming that alkaline hydrolysis of RNA results in about equal amounts of 2’- and 3’-mononucleotides, radioactivity in both regions was obtained by doubling that in 3’-GMP. Recovery of marker 14C!-labeled 2’- and 3’GMP added to unlabeled oocytes after KOH hydrolysis was about 70%, and data points correspondingly were corrected. In five experiments of this type, the mean rates of RNA synthesis (see later) calculated from 2’,3’-GMP were within 12% of the mean values derived from acid-precipitable material in the corresponding oocytes which were cornbusted. d. Rate Calculations and Measurement Precursor pool Specific Activity
of
Rates of RNA synthesis were calculated from the kinetics of incorporation of radioactivity into RNA as described (LaMarca et al., 1973). Pool specific activities at several time points in oocytes incubated in [3H]guanosine were determined after separation of nucleotides by chromatography on PEI-cellulose as described (LaMarca et al., 1973) except that spots occasionally were eluted with MgCl,-Tris buffer (Neuhard et al., 1965). Radioactivity in ATP averaged 3.7 and 3.0% of that in GTP in control and maturing oocytes, respectively, and no radioactivity was found in CTP and UTP. Since the ATP pool also is much larger than GTP (Woodland and Pestell, 1972), we ignored the contribution of radioactive ATP in calculating rates of synthesis. In some experiments in which 13HlGTP was injected into oocytes, specific activity of the GTP pool also was determined, as above, but only at two time-points (0 and 6 hr after injection). We found that in both control and maturing oocytes of four different animals, the specific activity of GTP decreased an average of less than 7% during the 6-hr period, and we used the mean of the initial and final values in calculations. In most experiments, GTP specific
LAMARCA et al.
Hormonal
Effects
on Oocyte RNA
387
Synthesis
oocytes from unstimulated and stimulated females exhibit similar uptake rates; linear uptake was observed throughout the entire incubation period. Likewise, the data show clearly that progesterone has no immediate effect on uptake of acid-soluble radioactivity, at least in oocytes from unstimulated females. Maturing oocytes dis-
activity was estimated by taking the amount of injected radioactive GTP, adjusting it for the average reduction during the incubation period, and dividing this by the average endogenous pool size (LaMarca et al., 1973). In oocytes of four unstimulated animals, the mean GTP pool size was 267.2 f. 67.6 pmole and maturing oocytes did not have significantly different pool sizes at 6 or 12 hr after progesterone exposure. Oocytes from four animals that had been induced to ovulate at times varying from 3 to 46 days previously had a mean GTP pool size of 272.8 + 29.1 pmoles. As with oocytes from unstimulated animals, the size of the GTP pools in maturing oocytes was not significantly different from that in controls.
45-40-
3 0-355 LJ30v 0 0 P
25. zo15-
RESULTS
a. Rates of RNA Synthesis in Incubated Oocytes An example of the kinetics of uptake of exogenous guanosine by control and maturing oocytes, taken from unstimulated females, is shown in Fig. 1. Data on the actual rate of uptake are summarized in Table 1, along with data on control oocytes from stimulated females. While there is substantial variability in oocytes from different females, the data show that control
FIG. 1. Uptake of total PCA-soluble radioactive material by oocytes incubated in 100 &i/ml r3Hlguanosine; each point represents average of 10 oocytes; (-) control oocytes; (----) maturing oocytes were exposed to progesterone (5 pg/ml) for 10 min immediately preceding incubation in [3HIguanosine. The lines were fit to the data by linear least squares analysis. Maturing oocytes underwent GVBD 5-7 hr after exposure to progesterone.
TABLE
1
RATE OF UFTAKE OF TOTAL PCA-SOLUBLE RADIOACTIVE MATERIAL” Type female
Unstimulated
Stimulated
Animal
1 2 3 4 5 6 7 8
Control
3659.2 397.0 1673.8 445.0 1682.0 525.0 817.0 1346.0
oocytes
+ 28.6 9.1 -t + 67.6 k 25.0 c 101.0 r 27.0 k 88.0 + 138.0
Maturing
oocytesb
Before GVBD
After
3577.0 k 35.5 435.4 2 16.3 1603.2 + 8.02 -
388.5 k 74.6 36.4 k 21.9 176.4 ” 25.6 -
GVBD
a Data in these experiments obtained by incubation of oocytes in 100 &i of 13H]guanosine; values are expressed in disintegrations per minute per oocyte per hour; Confidence intervals ? 1 SD (a) of the slope of the line fit to the incorporation data (Fig. 1) by linear least-squares analysis. b Maturing oocytes exposed to progesterone (5 @g/ml) for 10 min immediately preceding incubation in [3Hlguanosine.
388
DEVELOPMENTAL BIOLOGY
played linear uptake prior to GVBD which was indistinguishable from that of controls. After GVBD, there was an abrupt decrease in the rate of uptake, to about 10% of the previous value. Since oocytes first incubated with guanosine after GVBD also exhibited restricted uptake, this decrease apparently reflects the change in permeability coincident with GVBD already observed with respect to other exogenous materials (Ecker and Smith, 1971). Because of this reduced uptake, however, we have restricted rate measurements in incubated oocytes to time periods prior to GVBD. The specific activity of the GTP pool in oocytes incubated with guanosine also increases linearly, both in control and maturing oocytes (Fig. 2A). Figure 2B shows the kinetics of incorporation of radioactive precursor into RNA. As described previously (LaMarca et al., 19731, parabolas of this type fit the equation,
VOLUME 41, 1975
Z = (RK/2)tZ, in which the actual rate of incorporation CR)of GTP into stable RNA may be calculated from the incorporation constant (RK/2) of the curve which best fits the observed incorporation data (1) and the rate of increase (K) of the GTP specific activity. In experiments with oocytes from two-different unstimulated females (Table 21, progesterone-exposed oocytes prior to GVBD synthesized and accumulated stable RNA at rates demonstrably higher than in corresponding control oocytes.
b. Rates of RNA Synthesis in Injected Oocytes As indicated above, the time available for uptake of exogenous radioactivity is relatively short for maturing oocytes from unstimulated females (6-9 hr). It is prohibitively short in oocytes from stimulated females, which can undergo GVBD within s
(t’ - 600
I’ /’ 4 /
8
I
I
2
3
I 4
( 5
, 6
I 7
I 6
- 500
, 9
HOURS
FIG. 2. (A) GTP specific activities of oocytes incubated in 100 &i/ml 13Hlguanosine; each point calculated from the GTP extracted from 100 incubated oocytes and 200 “carrier” oocytes; (--) control oocytes; (----) maturing oocytes which were exposed to progesterone (5 ~glml) for 10 min immediately preceding incubation in 13H1guanosine; the lines were fit to the data by linear least-squares analysis. (B) Incorporation of radioactivity into PCA-precipitable, alkali-soluble material by control oocytes (--) and maturing oocytes f----l incubated in 100 &i/ml [3H]guanosine. Each point represents the mean value for 10 oocytes. Curves are parabolas of the type I = (RK/2)t2, which were fit to the data by least-squares analysis. Oocytes for both (Al and (B) are from same animal, and maturing oocytes underwent GVBD at 0 hr.
LAMARCA TABLE
et al.
Hormonal
Effects
2
RATES OF INCORPORATION OF GTP INTO STABLE RNA” Animal
Control oocytesb
Maturing oocytesb
Maturing/controlc
1 2
1.31 k 0.11 1.41 2 0.07
2.23 2 0.11 2.18 k 0.20
1.70 2 0.17 1.55 k 0.16
on Oocyte RNA
Synthesis
389 0 /
a Data obtained by incubation of oocytes in 100 &i/ml solutions of 13H1guanosine; neither animal had been previously stimulated with HCG. * Values are expressed as picomoles of GTP incorporated per hour per oocyte. c Ratios of corresponding rates. All uncertainty intervals above are + o which were calculated from the o of the constant (RK/2) of the parabolas fit to the incorporation data (Fig. 2B) and the (T of the slopes (K) of the lines lit to the data showing increase in GTP specific activities (Fig. 2A).
2 hr of progesterone exposure (Reynhout et al., 1975). Hence, most experiments on rates of RNA synthesis were performed by injecting GTP directly into oocytes. An example of the kinetics of incorporation of injected ll*CIGTP into the RNA of control and maturing oocytes is shown in Fig. 3. Identical results have been obtained after injection of oocytes with 13HlGTP. The kinetics of incorporation are more complex than was indicated in the two experiments reported earlier (LaMarca et al., 1973). In that case, we fit a single linear function to the data, even though incorporation values for the early time points were somewhat high, assuming that newly synthesized RNA was stable over the time course of the experiments. It now is clear that incorporation is biphasic, both in control and maturing oocytes, indicating the synthesis of both stable and unstable RNA species. Independent experiments verifying this are in progress, and will be presented subsequently including accurate measurements of synthetic and degradation constants (Anderson, Dolecki and Smith, in preparation). For the present, estimates of the initial rate of synthesis have been obtained by
FIG. 3. Incorporation of [WIGTP into PCA-precipitable material by oocytes injected with 60,400 dpm [WIGTP (500 mCiimmole1; each point represents the amount “incorporated” by three oocytes in zero time; (--) control oocytes; (----) maturing oocytes exposed to progesterone (5 pg/ml) for 10 min immediately preceding injection. Lines fit to data by linear least-squares analysis.
fitting a linear function to data points over the first 60-90 min. Since this period would include RNA that already has begun to turn over, such estimates are minimal for total RNA synthesis. A second linear function fit to data points beyond 90 min represents largely the rate of synthesis of stable RNA, including rRNA, since the contribution of radioactivity from unstable RNA at these time points becomes progressively less significant. The data in Table 3 summarize results obtained on oocytes from 11 unstimulated females. In control oocytes, the mean rate of synthesis of stable RNA (1.62 pmole/hr/oocyte) is consistent with the range of values measured in this paper by incubation of oocytes in 13Hlguanosine (Table 2). The latter, because of the absence of early time points, includes almost exclusively stable RNA. In contrast, the initial rate of synthesis (total RNA, Table 3)
390
DEVELOPMENTAL BIOL~CY TABLE RATES OF INCORPORATION OF GTP INTO RNA”
Animal
1 2 3 4 5 6 7 8 9 10 11 Mean ? SE”
Control
3.95 5.68 3.47 4.57 2.89 5.26 3.56 3.12 3.60 3.03 3.27 3.88
2 + + + + + 2 ? t ? ? 2
1.03” 1.46 0.93 1.38 0.48 1.51 0.88 0.78 0.91 0.77 0.84 0.31
oocytes*
2.10 2.24 1.92 1.40 1.18 2.15 1.56 0.82 2.01 1.36 0.97 1.62
2 2 + k * k + + & 2 + +
VOLUME 47, 1975 3
IN O~CYTES FROM UNSTIMULATED FEMALES
Maturing
0.57” 0.60 0.49 0.36 0.10 0.22 0.48 0.20 0.54 0.38 0.25 0.17
4.12 5.81 3.31 4.81 2.67 5.54
?z 1.03e 2 1.47 ?z 0.85 k 1.27 -c 0.22 2 0.46 4.38 -c 0.51
oocytes*
3.38 3.27 2.69 2.24 1.87 3.14
+ k 2 2 2 2
Maturing/control’
0.97e 0.83 0.71 0.65 0.15 0.33
2.77 2 0.25
1.04 1.02 0.95 1.05 0.92 1.05
2 + + k k + -
0.08 0.08 0.10 0.20 0.13 0.07
1.61 1.46 1.40 1.60 1.59 1.46
+ + f k + k -
0.28 0.15 0.14 0.24 0.08 0.15
a Data in these experiments obtained by microinjection of radioactive GTP into oocytes. * Values are expressed as picomoles of GTP incorporated per hour per oocyte. c Total rates of RNA synthesis calculated from “initial slope of incorporation kinetics line (see text). Experiments 5-7 were obtained by injecting oocytes with [‘4C]GTP. All others involve YHIGTP. d Rates of stable RNA synthesis calculated from “second” slope of kinetics line (see text). e Uncertainty intervals are 2 c which are calculated from the o of the slopes of the lines tit to the incorporation data (Fig. 2) and the (T of the average GTP pool size. f Ratios of corresponding rates; uncertainty intervals are + (T which were calculated in each experiment from (T of the slope of the two lines fit to the incorporation data. g SE. standard error of mean of the calculated rates. In all experiments, lines were fit to the data by leastsquares analysis.
clearly is much higher, ranging from 1.8 to 3.3 x the rate of stable RNA transcription. The pattern of RNA synthesis in control oocytes from stimulated females (Table 4) is essentially the same as in oocytes from unstimulated females; ratios between synthetic rates for total RNA compared to stable RNA range from 1.5 to 3.2 X . However, the mean rates of synthesis of both total RNA and stable RNA are significantly higher (1.37 and 1.45x, respectively) than the corresponding values for control oocytes from unstimulated females (Table 3). As indicated in Fig. 3, exposure of oocytes from unstimulated females to progesterone produced no measurable effect on initial rates of synthesis (Table 3). However, consistent with the incubation experiments (Table 21, maturing oocytes, prior to GVBD, synthesized stable RNA at 1.4-1.6 times the rate in control oocytes. In matur-
ing oocytes from HCG-stimulated animals, GVBD occurs too rapidly to be able to measure rates of synthesis of stable RNA prior to nuclear dissolution. However, initial rates of synthesis are no different in oocytes exposed to progesterone than in comparable control oocytes (Table 4). After GVBD, radioactivity in acid precipitable material still is detectable. However, because of extreme variability at individual time points in the level of radioactivity, frequently less than zero time point, rate calculations have been inconclusive. In fact, in only 2 of 11 experiments involving oocytes exhibiting GVBD (data not shown) from both stimulated and unstimulated females was a calculated rate of synthesis greater than corresponding errors. Both values were less than 5% of the values seen in corresponding oocytes prior to GVBD. Since mitochondria account for l-
LAMARCA et al.
Hormonal
Effects
TABLE RATES OF INCORPORATION OF GTP INTO RNA” Animal
Control Total RNA” 3 3 17
4 5h 6 7 Mean 2 SE*
22 46 52 54
6.09 (5.85 4.61 (4.85 5.56 (4.80 5.74 4.34 (5.19 5.48 5.43 5.33
+ k 2 2 -t + e t k + 2 k
on Oocyte RNA
4
IN O~CYTES FROM HCG-STIMULATED Maturing oocytes*
oocytes Stable RNAd
.40 .73) .54 .77) .34 59) .66 .58 .89) .63' .59' .15
391
Synthesis
1.90 (1.83 1.56 (1.65 1.99 (1.72 2.78 2.89 (3.46 2.57 2.79 2.40
+ k + + 2 + + -+ + + + 2
.04 20) .06 ,191 .25 .28) .32" .43 .62) .45' .41' .26
FEMALES
Maturing/control’
Total RNA’ 6.06 (5.42 4.51 (5.11 5.41 (4.78 5.58 4.19 (5.27 5.26
2 + + 2 t k + 2 k 2
.61 .79) .26 .62) .42 .62) .61' .31 .68) .60
-
1.00 (0.93 0.98 (1.05 0.97 (1.00 0.97 0.97 (1.02 0.96
lr + + + 2 t + + 2 k
.12 .ll) .13 .14) .lO .lO) .05 .14 .15) .06
-
5.24 k .ll
a***c*d*ef*oSame as in Table 3, except all experiments involved injection of VH]GTP. h Experiments in which rates were calculated using measured GTP specific activities; uncertainty intervals on rates k o, calculated from o of slopes of lines tit to incorporation data. The numbers in parentheses represent values calculated from average GTP pool size (Materials and Methods) for comparison.
2% of the RNA synthesized prior to GVBD and remains relatively constant through the completion of maturation, it seems likely that incorporation measured in intact oocytes after GVBD represents largely mitochondrial RNA synthesis (see Webb et al., 1975). DISCUSSION
Early indications that HCG might alter rates of RNA synthesis were based on the observation that incorporation of radioactive precursors into RNA was much greater in “large” oocytes remaining in the ovaries after the induction of ovulation than in similar sized preovulatory oocytes. (Davidson et al., 1964; Brown and Littna, 1964). Mairy and Denis (1971) provided the first direct comparison of levels of incorporation in various size classes of oocytes, maintained in vitro, taken from nonstimulated and HCG-stimulated females. Their data were consistent with the interpretation that HCG preferentially stimulated incorporation into “large” oocytes, although they pointed out that the results could also result simply from increased up-
take of precursor into such oocytes. In this connection, Hallberg and Smith (1975) have observed that, in uitro, oocytes taken from females injected with HCG had higher rates of amino acid uptake and incorporation into acid precipitable material relative to oocytes from non-hormonetreated females. Our data on rates of uptake of exogenous guanosine by different females (Table 1) show obvious overlap when stimulated and unstimulated females are compared. On the other hand, we have not compared rates of uptake in oocytes from the same female before and after treatment with HCG. Considering also the variability in rates of uptake, both in stimulated and unstimulated animals, the results do not allow complete rejection of the idea that ooctyes from stimulated females take up precursor more rapidly than comparable oocytes from unstimulated females. However, this question becomes less critical in studies involving actual measurement of RNA synthetic rates. A comparison of the data for “stimulated” females (Table 4) with that for “un-
392
DEVELOPMENTALBIOLOGY
VOLUME 47, 1975
stimulated” females (Table 3) shows that all do not support the idea that HCG results in any substantial change in rates of the former exhibit mean rates of synthesis that are higher than the latter, both for RNA synthesis. total RNA (37%) and stable RNA (48%). Concerning possible steroid effects, The significance of these differences is un- there is no indication from our data (Tacertain, however, largely because rates of bles 3 and 4) that the induction of maturaRNA synthesis listed in Table 3 vary sub- tion with progesterone alters the initial stantially (almost threefold for stable rate of RNA synthesis. However, progesRNA), and the higher values are well terone clearly results in an increase in the within the range of values obtained with rate of synthesis of stable RNA, measured oocytes from stimulated females. The both by injection of oocytes with [3H]GTP source of this variability is not known but (Table 3) and by incubation of oocytes with could relate partly to the unknown physio13Hlguanosine (Table 2). In theory, this logical states of the animals collected in observation could be explained by assumthe wild (see Wallace and Bergink, 1974). ing that, in response to progesterone, The data summarized in Table 4 indi- there is an increase in the rate of synthesis cate no significant difference in rates of of stable RNA species with a correspondRNA synthesis in oocytes taken from fe- ing decrease in the synthesis of unstable males at times ranging from 3 to 54 days RNA. Alternatively, progesterone could reafter injection with HCG. This time span sult in increased stability of certain RNA is thought to be significant for two rea- species which, in control oocytes, were less sons. First, pinocytotic uptake (in vitro) of stable. Either suggestion implies a qualitathe yolk precursor protein vitellogenin is tive change in RNA synthesis during matustimulated in oocytes taken from females ration. In the present study, we have not injected with HCG (see Wallace and Ber- attempted a careful qualitative study of RNA synthesized in control and maturing gink, 1974). However, enhanced uptake was found to persist for only about 3-4 oocytes, prior to germinal vesicle breakstudy (Anweeks after a single injection of the gonado- down. However, a preliminary tropin, suggesting, at least in this case, derson and Smith, unpublished data), indicates that, after a 2-hr pulse the percentthat HCG effects have dissipated within about 30 days. Second, according to Du- age of the total RNA migrating as rRNA on polyacrylamide gels is greater in maturing mont (1972), about 5-7 weeks is required for the reappearance of a population of than in control oocytes. The studies on RNA synthesis in full white banded oocytes in ovaries depleted of grown oocytes presented here indicate that these oocytes by the induction of ovulation with HCG. Again, this observation sug- the oocyte nucleus, up to the point of dissolution during maturation, continues to gests that HCG effects on oocyte growth function as an “oogenesis” nucleus, i.e., at have been completed within 35-49 days. pattern of RNA synIt is not certain that potential HCG ef- least the quantitative fects on RNA synthesis also would have thesis closely resembles that seen during dissipated within the time periods dis- oocyte growth (LaMarca et al., 1973). Howcussed above. If not, comparison of the ever, the necessity and possible functions data in Table 3 versus Table 4 would sup- of RNA synthesized during maturation remain unclear; RNA synthesis during this port the idea that HCG results in a stimutime is not required for the immediate lation of RNA synthetic rates. However, nor apparently for based on the above discussion, we suggest events of maturation develthat Animals 5-7 in Table 4 are more anal- the initial phases of post fertilization ogous to unstimulated than stimulated fe- opment (review by Smith 1975). In fact, males. Thus, we suggest that the data over- even the continued synthesis of RNA in
LAMARCA et al.
Hormonal
full grown control oocytes has evoked some uncertainty. Colman (1974) has suggested several alternatives, including “considerable” turnover of RNA, to explain the apparent anomaly that full grown oocytes contain only about 4 pg of RNA, yet appear to synthesize stable RNA continuously, which RNA should accumulate. This anomaly is readily understood, however, if one simply assumes that the amount of RNA in full grown oocytes represents a steadystate level, maintained by continual synthesis and degradation. Detection of all unstable species would not necessarily be possible in the relatively short term experiments presented here, particularly in cases (e.g. rRNA) in which radioactive molecules enter a large “pool” of unlabeled molecules. In this context, the recent study of Leonard and LaMarca (1975) reports that the half-life of rRNA in oocytes from different Xenopus laevis varies from 9 to 30 days. REFERENCES BENNETT, G. N., and GILHAM P. T. (1975). “Single addition” substrates for the synthesis of specific oligoribonucleotides with polynucleotide phospho2’-0-(X-methoxySynthesis of rylase. ethyllnucleoside 5’-diphosphates. Biochemistry, in press. BROWN, D. D., and LITTNA, E. (1966). RNA synthesis during the development of Xenopus laevis, the South African clawed toad. J. Mol. Biol. 20: 81-94. COLMAN, A. (1974). Synthesis of RNA in oocytes of Xenopus laevis during culture in vitro. J. Embryol. Exp. Morphol. 32, 515-532. DAVIDSON, E. H., CRIPPA, M., KRAMER, F. R., and MIR~KY A. E. (1964) Genomic function during the lampbrush chromosome stage of amphibian oogenesis. Proc. Nat. Acad. Sci. USA 56, 856863. DUMONT, J. N. (1972). Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals. J. Morphol. 136,153180. ECKER, R. E., and SMITH, L. D. (1971). Influence of
Effects
on Oocyte RNA
Synthesis
393
exogenous ions on the events of maturation in Rana pipiens oocytes. J. Cell. Physiol. 77, 61-70. EMERSON, C. P., and HUMPHREYS, T. (1970). A simple and sensitive method for quantitative measurment of cellular RNA synthesis. Anal. Biochem. 40, 254-266. HALLBERG, R. L., and SMITH, D. C. (19751 Ribosomal protein synthesis in Xenopus laevis oocytes. Develop. Biol. 42, 40-52. LAMARCA, M. J., SMITH, L. D., and STROBEL, M. (1973). Quantitative and qualitative analysis of RNA synthesis in stage 6 and stage 4 oocytes of Xenopus laevis. Develop. Biol. 34, 106-118. LEONARD, D. A., and LAMARCA, M. J. (1975). In vivo synthesis and turnover of cytoplasmic ribosomal RNA by stage 6 oocytes of Xenopus laevis. Develop. Biol., in press. MAIRY, M., and DENIS, H. (19711. Recherches biochimiques sur l’oogenese. I. Synthese du crapaud Sud African Xenopus laevis. Develop. Biol. 24, 143165. NEUHARD, J., RANDERATH, E., and RANDERATH, K. (1965). Ion exchange thin-layer chromatography. XIII. Resolution of complex nucleoside triphosphate mixtures. Anal. Biochem. 13, 211-222. REYNHOUT, J. K., TADDEI, C., SMITH, L. D., and LAMARCA, M. J. (1975). Response of large oocytes of Xenopus laevis to progesterone in vitro in relation to oocyte size and time after previous HCGinduced ovulation. Develop. Biol. 44, 375-379. SMITH, L. D. (19751. Molecular events during oocyte maturation. In “Biochemistry of Animal Development” (R. Weber, ed.), Vol. 3, pp. l-46. Academic Press, New York. WALLACE, R. A., and BERGINK, E. W. (1974). Amphibian vitellogenin: Properties, hormonal regulation of hepatic synthesis and ovarian uptake and conversion to yolk proteins. Amer. 2001. 14,11591176. WASSARMAN, W. J., and MASUI, Y. (1974). A study of gonadotropin action in the induction of oocyte maturation in Xenopus laevis. Biol. Reprod. 11, 133144. WEBB, A. C., LAMARCA, M. J., and SMITH, L. D. (1975). Synthesis of mitochondrial RNA by full grown and maturing oocytes of Rana pipiens and Xenopus laevis. Develop. Biol. 45, 44-55. WOODLAND, H. R., and PESTELL, R. Q. W. (1972). Determination of the nucleoside triphosphate contents of eggs and oocytes of Xenopus laevis. Biothem. J. 127, 597-605.
Hormonal
BIOLOGY
47, 384-393 (19%)
Effects on RNA Synthesis by Stage 6 Oocytes laevis l
MICHAEL J. LAMARCA*, MARJORIE C. STROBEL FIDLER, L. DENNIS Department
of Biological
KIRSTEN Sciences, Purdue
KEEM University
West Lafayette,
Indiana
of Xenopus
SMITH,
AND
47907
Accepted June 26,1975 RNA synthesis has been studied in “large” oocytes of Xenopus laevis, both as a function of time after injection of females with human chorionic gonadotropin (HCG) and in relation to the induction of maturation with progesterone in vitro. Rates of RNA synthesis were measured by analyzing the kinetics of incorporation of exogenous 13Hlguanosine, and microinjected [3H]- or [*%IGTP, into acid-precipitable material, coupled with measurements of precursor pool specific activity. The kinetics of incorporation into RNA of injected precursor are biphasic, indicating the synthesis of both stable and unstable RNA species. Estimates of the total rate of synthesis (stable and unstable) were derived from fitting a linear function to data over the first 60-90 min, while a linear function fit to the data beyond 90 min represented largely the synthesis of stable RNA species. Exposure of oocytes to progesterone had no effect on initial synthetic rates, but maturing oocytes synthesized stable RNA at 1.4-1.6 times the rate in control oocytes. A comparison of data obtained with oocytes from unstimulated (no prior HCG treatment) and HCG-stimulated females indicated that HCG has no substantial effect on rates of RNA synthesis. The signilicance of continued RNA synthesis in large full grown oocytes is discussed. INTRODUCTION
It now appears certain that amphibian oocytes remain synthetically active as they complete their growth phase, both in terms of protein and RNA synthesis (review by Smith, 1975). Studies on protein synthesis, primarily with Rana pipiens oocytes, further demonstrated that the level of synthetic activity is under hormonal control, especially during the period of oocyte maturation (see Smith, 1975). Similar studies on RNA synthesis, almost exclusively with Xenopus laevis oocytes, have been incomplete and somewhat contradictory. Brown and Littna (1966) reported that during the period of ovulation, Xenopus Zaevis oocytes synthesized nanogram quani Research supported by grants from the USPHS (No. HD04229) and the National Science Foundation (No. GB39971). This work was initiated while the principal investigator was an NSF. Science Faculty Fellow in the Department of Biological Sciences, Purdue University, West Lafayette, Ind. 47907. 2 Present address: Department of Biology, Lawrence University, Appleton, Wis. 64911. 384 Copyright 0 1975 by Academic Ress, Inc. All rights of reproduction in any form reserved.
tities of DNA-like RNA, presumably in response to the gonadotropin (HCG) used to induce ovulation. Subsequently, Mairy and Denis (1971) reported that large oocytes (1.0-1.2 mm diameter) taken from females recently induced to ovulate with HCG incorporated greater amounts of exogenous guanosine into RNA than similarsized oocytes taken from females not treated with HCG. However, potential permeability changes in response to the hormone were not monitored, and increased incorporation could have resulted from greater uptake of precursor in oocytes from HCG-treated females (see Mairy and Denis, 1971). Recently, both uptake and incorporation of radioactive precursors into RNA have been measured in ovarian follicles removed from HCG-stimulated females (Colman, 1974) and in ovarian tissue exposed to HCG or progesterone in vitro (Wassarman and Masui, 1974). Neither parameter was obviously altered during long-term (24 hr) or shorter (6 hr) pulses to radioactive precursors, but the differential behavior of oocyte versus so-
LAMARCA et al.
Hormonal
matic follicular components was inadequately quantitated. Recently, we studied RNA synthesis in the largest oocytes taken from Xenopus females that had not been induced to ovulate (LaMarca et al., 1973). Rates of RNA synthesis, determined from kinetics of guanosine (GTP) incorporation into RNA and changes in precursor pool specific activity, were at least as great in these large oocytes as in smaller oocytes (500-600 pm diameter). We now report similar experiments in which rates of RNA synthesis were measured in isolated “large” oocytes, both as a function of time after injection of females with HCG and in relation to the induction of maturation with progesterone in vitro. MATERIALS
AND
METHODS
a. Animals Mature Xenopus laevis females were obtained directly from South Africa and maintained as previously described (Webb et al., 1975). Animals classified as unstimulated refer to females collected directly from the wild and used after maintenance in the laboratory for varying time periods (minimally 4-6 weeks). They had not been induced to ovulate, and their ovaries contained white banded oocytes (see Dumont, 1972). Stimulated females had been induced to ovulate previously by injection of HCG (Antuitrin, Parke-Davis) into the dorsal lymph sac; 500 IU of HCG followed by an additional injection of 250 IU the following day. b. Preparation
of Oocytes
Ovarian lobes were removed from pithed females and were washed extensively in antibiotic Ringer’s solution as described (LaMarca et al., 1973). Oocytes were manually dissected from their follicles and adhering follicle cells removed by Pronase digestion (LaMarcaet al., 1973). All experiments were conducted on oocytes with diameters about 1.2 mm or greater. With
Effects
on Oocyte RNA
Synthesis
385
unstimulated females, the oocytes always contained white equatorial bands. After ovulation, white banded oocytes were no longer present and the remaining population of large oocytes, still about 1.2 mm in diameter, correspond more nearly to stage 5, as described by Dumont (1972). However, both these oocytes and the white banded ones respond similarly to progesterone (Reynhout et al., 1975). Maturation was induced by exposure to progesterone (5 pglml) for 10 min. Greater than 99% underwent GVBD, judged externally by the appearance of a circular whitish area in the animal hemisphere (Reynhout et al., 1975) and verified by dissection of selected oocytes. All operations were performed at a temperature of approximately 20°C. c. Uptake and Incorporation tive Precursor
of Radioac-
Follicle-free oocytes were incubated in sterile antibiotic Ringer’s solution containing 100 &i/ml of L3H]guanosine (NEN, tetrasodium salt, S3H, 6.5 Ci/mmole). At hourly intervals, groups of 10 oocytes were removed, rinsed, and either homogenized or soaked overnight in 0.5 N perchloric acid (PCA). Results were comparable with either procedure. Acid-soluble radioactivity represented total uptake. Radioactivity incorporated into RNA at each time point equaled the fraction of acid-insoluble radioactivity hydrolyzable by 0.3 N KOH as described (LaMarca et al., 1973). This procedure could overestimate rates of synthesis, since unincorporated nucleoside phosphates trapped in the acid-precipitable pellet would also be released by alkaline hydrolysis (see Emerson and Humphreys, 1970). However, results from these experiments are not significantly different from those obtained under conditions in which this problem has been taken into account. In most experiments, oocytes were injected directly with 15-20 nliters of 13HlGTP (NEN, tetrasodium salt, 5.28 Ci/mmole) or [%]GTP (Amersham-
386
DEVELOPMENTAL BIOLOGY
Searle, 500-545 mCi/mmole). At appropriate times after injection, oocytes were either homogenized in 0.5 iV PCA, followed by collection of precipitates on Millipore filters (HAWP, 45 pm pore) or were placed into cold 0.5 N PCA for overnight extraction of unincorporated material. Results with either procedure were essentially identical. In most experiments, filters or intact oocytes were combusted, without prior alkaline hydrolysis, and radioactivity incorporated into RNA at each time point was taken to be acid-precipitable radioactivity remaining after subtraction of acid-precipitable radioactivity present at zero time after microinjection (LaMarca et al., 1973). In selected experiments, injected oocytes were further hydrolyzed in alkali, and radioactivity in 2’,3’-GMP was determined. To avoid exchange of tritium with water during alkaline hydrolysis, oocytes were injected with [14ClGTP. At various times after injection, groups of 15 oocytes were placed into cold (4°C) 0.5 N PCA, extracted overnight and then divided into two groups. One group of five oocytes was combusted directly and counted. The remaining 10 oocytes (at each time point) were hydrolyzed in 0.3 N KOH for 18 hr at 37°C. The KOH-soluble material was collected, neutralized with sulfuric acid and, after addition of OD quantities of 2’- and 3’GMP and 5’-GTP, the sample was lyophilized. Individual samples were resuspended in water, and aliquots were spotted directly on Whatman 3MM paper. Nucleotides were separated by descending chromatography in a high salt solvent (solvent D, Bennett and Gilham, 1975)consisting of water (100 ml) and (NH,),SO, (40 g) adjusted to pH 8.5 with concentrated NH,OH. Relative to 5’-GTP, 2’- and 3’GMP migrated with R, values of 0.83 and 0.68, respectively. However, 5’-GMP also cochromatographed with 2’-GMP. Hence, to determine the fraction of radioactivity present only in 2’,3’-mononucleotides from RNA, the 3’-GMP spot (identified by
VOLUME 47, 1975
marker 3’-GMP) was cut out and counted directly in toluene-PPO. Assuming that alkaline hydrolysis of RNA results in about equal amounts of 2’- and 3’-mononucleotides, radioactivity in both regions was obtained by doubling that in 3’-GMP. Recovery of marker 14C!-labeled 2’- and 3’GMP added to unlabeled oocytes after KOH hydrolysis was about 70%, and data points correspondingly were corrected. In five experiments of this type, the mean rates of RNA synthesis (see later) calculated from 2’,3’-GMP were within 12% of the mean values derived from acid-precipitable material in the corresponding oocytes which were cornbusted. d. Rate Calculations and Measurement Precursor pool Specific Activity
of
Rates of RNA synthesis were calculated from the kinetics of incorporation of radioactivity into RNA as described (LaMarca et al., 1973). Pool specific activities at several time points in oocytes incubated in [3H]guanosine were determined after separation of nucleotides by chromatography on PEI-cellulose as described (LaMarca et al., 1973) except that spots occasionally were eluted with MgCl,-Tris buffer (Neuhard et al., 1965). Radioactivity in ATP averaged 3.7 and 3.0% of that in GTP in control and maturing oocytes, respectively, and no radioactivity was found in CTP and UTP. Since the ATP pool also is much larger than GTP (Woodland and Pestell, 1972), we ignored the contribution of radioactive ATP in calculating rates of synthesis. In some experiments in which 13HlGTP was injected into oocytes, specific activity of the GTP pool also was determined, as above, but only at two time-points (0 and 6 hr after injection). We found that in both control and maturing oocytes of four different animals, the specific activity of GTP decreased an average of less than 7% during the 6-hr period, and we used the mean of the initial and final values in calculations. In most experiments, GTP specific
LAMARCA et al.
Hormonal
Effects
on Oocyte RNA
387
Synthesis
oocytes from unstimulated and stimulated females exhibit similar uptake rates; linear uptake was observed throughout the entire incubation period. Likewise, the data show clearly that progesterone has no immediate effect on uptake of acid-soluble radioactivity, at least in oocytes from unstimulated females. Maturing oocytes dis-
activity was estimated by taking the amount of injected radioactive GTP, adjusting it for the average reduction during the incubation period, and dividing this by the average endogenous pool size (LaMarca et al., 1973). In oocytes of four unstimulated animals, the mean GTP pool size was 267.2 f. 67.6 pmole and maturing oocytes did not have significantly different pool sizes at 6 or 12 hr after progesterone exposure. Oocytes from four animals that had been induced to ovulate at times varying from 3 to 46 days previously had a mean GTP pool size of 272.8 + 29.1 pmoles. As with oocytes from unstimulated animals, the size of the GTP pools in maturing oocytes was not significantly different from that in controls.
45-40-
3 0-355 LJ30v 0 0 P
25. zo15-
RESULTS
a. Rates of RNA Synthesis in Incubated Oocytes An example of the kinetics of uptake of exogenous guanosine by control and maturing oocytes, taken from unstimulated females, is shown in Fig. 1. Data on the actual rate of uptake are summarized in Table 1, along with data on control oocytes from stimulated females. While there is substantial variability in oocytes from different females, the data show that control
FIG. 1. Uptake of total PCA-soluble radioactive material by oocytes incubated in 100 &i/ml r3Hlguanosine; each point represents average of 10 oocytes; (-) control oocytes; (----) maturing oocytes were exposed to progesterone (5 pg/ml) for 10 min immediately preceding incubation in [3HIguanosine. The lines were fit to the data by linear least squares analysis. Maturing oocytes underwent GVBD 5-7 hr after exposure to progesterone.
TABLE
1
RATE OF UFTAKE OF TOTAL PCA-SOLUBLE RADIOACTIVE MATERIAL” Type female
Unstimulated
Stimulated
Animal
1 2 3 4 5 6 7 8
Control
3659.2 397.0 1673.8 445.0 1682.0 525.0 817.0 1346.0
oocytes
+ 28.6 9.1 -t + 67.6 k 25.0 c 101.0 r 27.0 k 88.0 + 138.0
Maturing
oocytesb
Before GVBD
After
3577.0 k 35.5 435.4 2 16.3 1603.2 + 8.02 -
388.5 k 74.6 36.4 k 21.9 176.4 ” 25.6 -
GVBD
a Data in these experiments obtained by incubation of oocytes in 100 &i of 13H]guanosine; values are expressed in disintegrations per minute per oocyte per hour; Confidence intervals ? 1 SD (a) of the slope of the line fit to the incorporation data (Fig. 1) by linear least-squares analysis. b Maturing oocytes exposed to progesterone (5 @g/ml) for 10 min immediately preceding incubation in [3Hlguanosine.
388
DEVELOPMENTAL BIOLOGY
played linear uptake prior to GVBD which was indistinguishable from that of controls. After GVBD, there was an abrupt decrease in the rate of uptake, to about 10% of the previous value. Since oocytes first incubated with guanosine after GVBD also exhibited restricted uptake, this decrease apparently reflects the change in permeability coincident with GVBD already observed with respect to other exogenous materials (Ecker and Smith, 1971). Because of this reduced uptake, however, we have restricted rate measurements in incubated oocytes to time periods prior to GVBD. The specific activity of the GTP pool in oocytes incubated with guanosine also increases linearly, both in control and maturing oocytes (Fig. 2A). Figure 2B shows the kinetics of incorporation of radioactive precursor into RNA. As described previously (LaMarca et al., 19731, parabolas of this type fit the equation,
VOLUME 41, 1975
Z = (RK/2)tZ, in which the actual rate of incorporation CR)of GTP into stable RNA may be calculated from the incorporation constant (RK/2) of the curve which best fits the observed incorporation data (1) and the rate of increase (K) of the GTP specific activity. In experiments with oocytes from two-different unstimulated females (Table 21, progesterone-exposed oocytes prior to GVBD synthesized and accumulated stable RNA at rates demonstrably higher than in corresponding control oocytes.
b. Rates of RNA Synthesis in Injected Oocytes As indicated above, the time available for uptake of exogenous radioactivity is relatively short for maturing oocytes from unstimulated females (6-9 hr). It is prohibitively short in oocytes from stimulated females, which can undergo GVBD within s
(t’ - 600
I’ /’ 4 /
8
I
I
2
3
I 4
( 5
, 6
I 7
I 6
- 500
, 9
HOURS
FIG. 2. (A) GTP specific activities of oocytes incubated in 100 &i/ml 13Hlguanosine; each point calculated from the GTP extracted from 100 incubated oocytes and 200 “carrier” oocytes; (--) control oocytes; (----) maturing oocytes which were exposed to progesterone (5 ~glml) for 10 min immediately preceding incubation in 13H1guanosine; the lines were fit to the data by linear least-squares analysis. (B) Incorporation of radioactivity into PCA-precipitable, alkali-soluble material by control oocytes (--) and maturing oocytes f----l incubated in 100 &i/ml [3H]guanosine. Each point represents the mean value for 10 oocytes. Curves are parabolas of the type I = (RK/2)t2, which were fit to the data by least-squares analysis. Oocytes for both (Al and (B) are from same animal, and maturing oocytes underwent GVBD at 0 hr.
LAMARCA TABLE
et al.
Hormonal
Effects
2
RATES OF INCORPORATION OF GTP INTO STABLE RNA” Animal
Control oocytesb
Maturing oocytesb
Maturing/controlc
1 2
1.31 k 0.11 1.41 2 0.07
2.23 2 0.11 2.18 k 0.20
1.70 2 0.17 1.55 k 0.16
on Oocyte RNA
Synthesis
389 0 /
a Data obtained by incubation of oocytes in 100 &i/ml solutions of 13H1guanosine; neither animal had been previously stimulated with HCG. * Values are expressed as picomoles of GTP incorporated per hour per oocyte. c Ratios of corresponding rates. All uncertainty intervals above are + o which were calculated from the o of the constant (RK/2) of the parabolas fit to the incorporation data (Fig. 2B) and the (T of the slopes (K) of the lines lit to the data showing increase in GTP specific activities (Fig. 2A).
2 hr of progesterone exposure (Reynhout et al., 1975). Hence, most experiments on rates of RNA synthesis were performed by injecting GTP directly into oocytes. An example of the kinetics of incorporation of injected ll*CIGTP into the RNA of control and maturing oocytes is shown in Fig. 3. Identical results have been obtained after injection of oocytes with 13HlGTP. The kinetics of incorporation are more complex than was indicated in the two experiments reported earlier (LaMarca et al., 1973). In that case, we fit a single linear function to the data, even though incorporation values for the early time points were somewhat high, assuming that newly synthesized RNA was stable over the time course of the experiments. It now is clear that incorporation is biphasic, both in control and maturing oocytes, indicating the synthesis of both stable and unstable RNA species. Independent experiments verifying this are in progress, and will be presented subsequently including accurate measurements of synthetic and degradation constants (Anderson, Dolecki and Smith, in preparation). For the present, estimates of the initial rate of synthesis have been obtained by
FIG. 3. Incorporation of [WIGTP into PCA-precipitable material by oocytes injected with 60,400 dpm [WIGTP (500 mCiimmole1; each point represents the amount “incorporated” by three oocytes in zero time; (--) control oocytes; (----) maturing oocytes exposed to progesterone (5 pg/ml) for 10 min immediately preceding injection. Lines fit to data by linear least-squares analysis.
fitting a linear function to data points over the first 60-90 min. Since this period would include RNA that already has begun to turn over, such estimates are minimal for total RNA synthesis. A second linear function fit to data points beyond 90 min represents largely the rate of synthesis of stable RNA, including rRNA, since the contribution of radioactivity from unstable RNA at these time points becomes progressively less significant. The data in Table 3 summarize results obtained on oocytes from 11 unstimulated females. In control oocytes, the mean rate of synthesis of stable RNA (1.62 pmole/hr/oocyte) is consistent with the range of values measured in this paper by incubation of oocytes in 13Hlguanosine (Table 2). The latter, because of the absence of early time points, includes almost exclusively stable RNA. In contrast, the initial rate of synthesis (total RNA, Table 3)
390
DEVELOPMENTAL BIOL~CY TABLE RATES OF INCORPORATION OF GTP INTO RNA”
Animal
1 2 3 4 5 6 7 8 9 10 11 Mean ? SE”
Control
3.95 5.68 3.47 4.57 2.89 5.26 3.56 3.12 3.60 3.03 3.27 3.88
2 + + + + + 2 ? t ? ? 2
1.03” 1.46 0.93 1.38 0.48 1.51 0.88 0.78 0.91 0.77 0.84 0.31
oocytes*
2.10 2.24 1.92 1.40 1.18 2.15 1.56 0.82 2.01 1.36 0.97 1.62
2 2 + k * k + + & 2 + +
VOLUME 47, 1975 3
IN O~CYTES FROM UNSTIMULATED FEMALES
Maturing
0.57” 0.60 0.49 0.36 0.10 0.22 0.48 0.20 0.54 0.38 0.25 0.17
4.12 5.81 3.31 4.81 2.67 5.54
?z 1.03e 2 1.47 ?z 0.85 k 1.27 -c 0.22 2 0.46 4.38 -c 0.51
oocytes*
3.38 3.27 2.69 2.24 1.87 3.14
+ k 2 2 2 2
Maturing/control’
0.97e 0.83 0.71 0.65 0.15 0.33
2.77 2 0.25
1.04 1.02 0.95 1.05 0.92 1.05
2 + + k k + -
0.08 0.08 0.10 0.20 0.13 0.07
1.61 1.46 1.40 1.60 1.59 1.46
+ + f k + k -
0.28 0.15 0.14 0.24 0.08 0.15
a Data in these experiments obtained by microinjection of radioactive GTP into oocytes. * Values are expressed as picomoles of GTP incorporated per hour per oocyte. c Total rates of RNA synthesis calculated from “initial slope of incorporation kinetics line (see text). Experiments 5-7 were obtained by injecting oocytes with [‘4C]GTP. All others involve YHIGTP. d Rates of stable RNA synthesis calculated from “second” slope of kinetics line (see text). e Uncertainty intervals are 2 c which are calculated from the o of the slopes of the lines tit to the incorporation data (Fig. 2) and the (T of the average GTP pool size. f Ratios of corresponding rates; uncertainty intervals are + (T which were calculated in each experiment from (T of the slope of the two lines fit to the incorporation data. g SE. standard error of mean of the calculated rates. In all experiments, lines were fit to the data by leastsquares analysis.
clearly is much higher, ranging from 1.8 to 3.3 x the rate of stable RNA transcription. The pattern of RNA synthesis in control oocytes from stimulated females (Table 4) is essentially the same as in oocytes from unstimulated females; ratios between synthetic rates for total RNA compared to stable RNA range from 1.5 to 3.2 X . However, the mean rates of synthesis of both total RNA and stable RNA are significantly higher (1.37 and 1.45x, respectively) than the corresponding values for control oocytes from unstimulated females (Table 3). As indicated in Fig. 3, exposure of oocytes from unstimulated females to progesterone produced no measurable effect on initial rates of synthesis (Table 3). However, consistent with the incubation experiments (Table 21, maturing oocytes, prior to GVBD, synthesized stable RNA at 1.4-1.6 times the rate in control oocytes. In matur-
ing oocytes from HCG-stimulated animals, GVBD occurs too rapidly to be able to measure rates of synthesis of stable RNA prior to nuclear dissolution. However, initial rates of synthesis are no different in oocytes exposed to progesterone than in comparable control oocytes (Table 4). After GVBD, radioactivity in acid precipitable material still is detectable. However, because of extreme variability at individual time points in the level of radioactivity, frequently less than zero time point, rate calculations have been inconclusive. In fact, in only 2 of 11 experiments involving oocytes exhibiting GVBD (data not shown) from both stimulated and unstimulated females was a calculated rate of synthesis greater than corresponding errors. Both values were less than 5% of the values seen in corresponding oocytes prior to GVBD. Since mitochondria account for l-
LAMARCA et al.
Hormonal
Effects
TABLE RATES OF INCORPORATION OF GTP INTO RNA” Animal
Control Total RNA” 3 3 17
4 5h 6 7 Mean 2 SE*
22 46 52 54
6.09 (5.85 4.61 (4.85 5.56 (4.80 5.74 4.34 (5.19 5.48 5.43 5.33
+ k 2 2 -t + e t k + 2 k
on Oocyte RNA
4
IN O~CYTES FROM HCG-STIMULATED Maturing oocytes*
oocytes Stable RNAd
.40 .73) .54 .77) .34 59) .66 .58 .89) .63' .59' .15
391
Synthesis
1.90 (1.83 1.56 (1.65 1.99 (1.72 2.78 2.89 (3.46 2.57 2.79 2.40
+ k + + 2 + + -+ + + + 2
.04 20) .06 ,191 .25 .28) .32" .43 .62) .45' .41' .26
FEMALES
Maturing/control’
Total RNA’ 6.06 (5.42 4.51 (5.11 5.41 (4.78 5.58 4.19 (5.27 5.26
2 + + 2 t k + 2 k 2
.61 .79) .26 .62) .42 .62) .61' .31 .68) .60
-
1.00 (0.93 0.98 (1.05 0.97 (1.00 0.97 0.97 (1.02 0.96
lr + + + 2 t + + 2 k
.12 .ll) .13 .14) .lO .lO) .05 .14 .15) .06
-
5.24 k .ll
a***c*d*ef*oSame as in Table 3, except all experiments involved injection of VH]GTP. h Experiments in which rates were calculated using measured GTP specific activities; uncertainty intervals on rates k o, calculated from o of slopes of lines tit to incorporation data. The numbers in parentheses represent values calculated from average GTP pool size (Materials and Methods) for comparison.
2% of the RNA synthesized prior to GVBD and remains relatively constant through the completion of maturation, it seems likely that incorporation measured in intact oocytes after GVBD represents largely mitochondrial RNA synthesis (see Webb et al., 1975). DISCUSSION
Early indications that HCG might alter rates of RNA synthesis were based on the observation that incorporation of radioactive precursors into RNA was much greater in “large” oocytes remaining in the ovaries after the induction of ovulation than in similar sized preovulatory oocytes. (Davidson et al., 1964; Brown and Littna, 1964). Mairy and Denis (1971) provided the first direct comparison of levels of incorporation in various size classes of oocytes, maintained in vitro, taken from nonstimulated and HCG-stimulated females. Their data were consistent with the interpretation that HCG preferentially stimulated incorporation into “large” oocytes, although they pointed out that the results could also result simply from increased up-
take of precursor into such oocytes. In this connection, Hallberg and Smith (1975) have observed that, in uitro, oocytes taken from females injected with HCG had higher rates of amino acid uptake and incorporation into acid precipitable material relative to oocytes from non-hormonetreated females. Our data on rates of uptake of exogenous guanosine by different females (Table 1) show obvious overlap when stimulated and unstimulated females are compared. On the other hand, we have not compared rates of uptake in oocytes from the same female before and after treatment with HCG. Considering also the variability in rates of uptake, both in stimulated and unstimulated animals, the results do not allow complete rejection of the idea that ooctyes from stimulated females take up precursor more rapidly than comparable oocytes from unstimulated females. However, this question becomes less critical in studies involving actual measurement of RNA synthetic rates. A comparison of the data for “stimulated” females (Table 4) with that for “un-
392
DEVELOPMENTALBIOLOGY
VOLUME 47, 1975
stimulated” females (Table 3) shows that all do not support the idea that HCG results in any substantial change in rates of the former exhibit mean rates of synthesis that are higher than the latter, both for RNA synthesis. total RNA (37%) and stable RNA (48%). Concerning possible steroid effects, The significance of these differences is un- there is no indication from our data (Tacertain, however, largely because rates of bles 3 and 4) that the induction of maturaRNA synthesis listed in Table 3 vary sub- tion with progesterone alters the initial stantially (almost threefold for stable rate of RNA synthesis. However, progesRNA), and the higher values are well terone clearly results in an increase in the within the range of values obtained with rate of synthesis of stable RNA, measured oocytes from stimulated females. The both by injection of oocytes with [3H]GTP source of this variability is not known but (Table 3) and by incubation of oocytes with could relate partly to the unknown physio13Hlguanosine (Table 2). In theory, this logical states of the animals collected in observation could be explained by assumthe wild (see Wallace and Bergink, 1974). ing that, in response to progesterone, The data summarized in Table 4 indi- there is an increase in the rate of synthesis cate no significant difference in rates of of stable RNA species with a correspondRNA synthesis in oocytes taken from fe- ing decrease in the synthesis of unstable males at times ranging from 3 to 54 days RNA. Alternatively, progesterone could reafter injection with HCG. This time span sult in increased stability of certain RNA is thought to be significant for two rea- species which, in control oocytes, were less sons. First, pinocytotic uptake (in vitro) of stable. Either suggestion implies a qualitathe yolk precursor protein vitellogenin is tive change in RNA synthesis during matustimulated in oocytes taken from females ration. In the present study, we have not injected with HCG (see Wallace and Ber- attempted a careful qualitative study of RNA synthesized in control and maturing gink, 1974). However, enhanced uptake was found to persist for only about 3-4 oocytes, prior to germinal vesicle breakstudy (Anweeks after a single injection of the gonado- down. However, a preliminary tropin, suggesting, at least in this case, derson and Smith, unpublished data), indicates that, after a 2-hr pulse the percentthat HCG effects have dissipated within about 30 days. Second, according to Du- age of the total RNA migrating as rRNA on polyacrylamide gels is greater in maturing mont (1972), about 5-7 weeks is required for the reappearance of a population of than in control oocytes. The studies on RNA synthesis in full white banded oocytes in ovaries depleted of grown oocytes presented here indicate that these oocytes by the induction of ovulation with HCG. Again, this observation sug- the oocyte nucleus, up to the point of dissolution during maturation, continues to gests that HCG effects on oocyte growth function as an “oogenesis” nucleus, i.e., at have been completed within 35-49 days. pattern of RNA synIt is not certain that potential HCG ef- least the quantitative fects on RNA synthesis also would have thesis closely resembles that seen during dissipated within the time periods dis- oocyte growth (LaMarca et al., 1973). Howcussed above. If not, comparison of the ever, the necessity and possible functions data in Table 3 versus Table 4 would sup- of RNA synthesized during maturation remain unclear; RNA synthesis during this port the idea that HCG results in a stimutime is not required for the immediate lation of RNA synthetic rates. However, nor apparently for based on the above discussion, we suggest events of maturation develthat Animals 5-7 in Table 4 are more anal- the initial phases of post fertilization ogous to unstimulated than stimulated fe- opment (review by Smith 1975). In fact, males. Thus, we suggest that the data over- even the continued synthesis of RNA in
LAMARCA et al.
Hormonal
full grown control oocytes has evoked some uncertainty. Colman (1974) has suggested several alternatives, including “considerable” turnover of RNA, to explain the apparent anomaly that full grown oocytes contain only about 4 pg of RNA, yet appear to synthesize stable RNA continuously, which RNA should accumulate. This anomaly is readily understood, however, if one simply assumes that the amount of RNA in full grown oocytes represents a steadystate level, maintained by continual synthesis and degradation. Detection of all unstable species would not necessarily be possible in the relatively short term experiments presented here, particularly in cases (e.g. rRNA) in which radioactive molecules enter a large “pool” of unlabeled molecules. In this context, the recent study of Leonard and LaMarca (1975) reports that the half-life of rRNA in oocytes from different Xenopus laevis varies from 9 to 30 days. REFERENCES BENNETT, G. N., and GILHAM P. T. (1975). “Single addition” substrates for the synthesis of specific oligoribonucleotides with polynucleotide phospho2’-0-(X-methoxySynthesis of rylase. ethyllnucleoside 5’-diphosphates. Biochemistry, in press. BROWN, D. D., and LITTNA, E. (1966). RNA synthesis during the development of Xenopus laevis, the South African clawed toad. J. Mol. Biol. 20: 81-94. COLMAN, A. (1974). Synthesis of RNA in oocytes of Xenopus laevis during culture in vitro. J. Embryol. Exp. Morphol. 32, 515-532. DAVIDSON, E. H., CRIPPA, M., KRAMER, F. R., and MIR~KY A. E. (1964) Genomic function during the lampbrush chromosome stage of amphibian oogenesis. Proc. Nat. Acad. Sci. USA 56, 856863. DUMONT, J. N. (1972). Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals. J. Morphol. 136,153180. ECKER, R. E., and SMITH, L. D. (1971). Influence of
Effects
on Oocyte RNA
Synthesis
393
exogenous ions on the events of maturation in Rana pipiens oocytes. J. Cell. Physiol. 77, 61-70. EMERSON, C. P., and HUMPHREYS, T. (1970). A simple and sensitive method for quantitative measurment of cellular RNA synthesis. Anal. Biochem. 40, 254-266. HALLBERG, R. L., and SMITH, D. C. (19751 Ribosomal protein synthesis in Xenopus laevis oocytes. Develop. Biol. 42, 40-52. LAMARCA, M. J., SMITH, L. D., and STROBEL, M. (1973). Quantitative and qualitative analysis of RNA synthesis in stage 6 and stage 4 oocytes of Xenopus laevis. Develop. Biol. 34, 106-118. LEONARD, D. A., and LAMARCA, M. J. (1975). In vivo synthesis and turnover of cytoplasmic ribosomal RNA by stage 6 oocytes of Xenopus laevis. Develop. Biol., in press. MAIRY, M., and DENIS, H. (19711. Recherches biochimiques sur l’oogenese. I. Synthese du crapaud Sud African Xenopus laevis. Develop. Biol. 24, 143165. NEUHARD, J., RANDERATH, E., and RANDERATH, K. (1965). Ion exchange thin-layer chromatography. XIII. Resolution of complex nucleoside triphosphate mixtures. Anal. Biochem. 13, 211-222. REYNHOUT, J. K., TADDEI, C., SMITH, L. D., and LAMARCA, M. J. (1975). Response of large oocytes of Xenopus laevis to progesterone in vitro in relation to oocyte size and time after previous HCGinduced ovulation. Develop. Biol. 44, 375-379. SMITH, L. D. (19751. Molecular events during oocyte maturation. In “Biochemistry of Animal Development” (R. Weber, ed.), Vol. 3, pp. l-46. Academic Press, New York. WALLACE, R. A., and BERGINK, E. W. (1974). Amphibian vitellogenin: Properties, hormonal regulation of hepatic synthesis and ovarian uptake and conversion to yolk proteins. Amer. 2001. 14,11591176. WASSARMAN, W. J., and MASUI, Y. (1974). A study of gonadotropin action in the induction of oocyte maturation in Xenopus laevis. Biol. Reprod. 11, 133144. WEBB, A. C., LAMARCA, M. J., and SMITH, L. D. (1975). Synthesis of mitochondrial RNA by full grown and maturing oocytes of Rana pipiens and Xenopus laevis. Develop. Biol. 45, 44-55. WOODLAND, H. R., and PESTELL, R. Q. W. (1972). Determination of the nucleoside triphosphate contents of eggs and oocytes of Xenopus laevis. Biothem. J. 127, 597-605.