Studies on Maturation in Xenopus laevis Oocytes I I I. Energy Production and Requirements for Protein Synthesis J. BRACHET, A. PAYS-DE SCHUTTER and E. HUBERT Laboratory of molecular Cytology and Embryology, Department of molecular Biology, University of Brussels, Belgium and Laboratorio di Embriologia moleculare C.N.R.Arco Felice, Napoli, Italy
Received February 197.5
The following results were obtained: 1) The oxygen consumption of progesterone-stimulated X . laevis oocytes increases at the time of germinal vesicle (GV) breakdown. 2) Continuous treatment with 1 mM KCN, 1 mM and 0.1 mM DNP completely inhibits GV breakdown. 3) Pretreatment experiments with KCN, DNP and cycloheximide show that binding of progesterone to the plasma membrane and the specific hormone receptor requires neither energy, nor protein synthesis. 4) 1 h pulses of DNP (1 mM) or cycloheximide (50 pg/ml) were applied, at various time intervals after progesterone treatment: early pulses strongly delay, but do not prevent GV breakdown; late pulses just before GV breakdown induce a number of cytological abnormalities, which ultimately lead to cytolysis. The significance of these results is discussed and the hypothesis that synthesis of the maturation producing factor (MPF) requires both energy and protein synthesis is proposed. Introduction
Meiotic maturation, in amphibian oocytes, has been the subject of several recent reviews [ l , 2, 3,41. Addition of progesterone or certain other steroid hormones to large oocytes induces breakdown of the large germinal vesicle (VG) and expulsion of the 1st polar body. Maturation can be subdivided into a number of steps: 1) Progesterone must first react with the plasma membrane, since it is inactive if injected into oocytes. 2) The hormone binds to a receptor, which is associated with pigment granules (melanosomes) [ 5 . 61. 3) It then induces the formation of several factors in the oocyte. The most important one ofthese is the maturation promoting factor (MPF): if cytoplasm taken from a progesterone-treated oocyte is injected into a normal oocyte, maturation is induced in the latter [6, 7,8,9, 10, 111. Homogenates from hormone-stimulated oocytes also contain a pseudomaturation inducing factor (PIF): if such homogenates are injected into normal oocytes, they undergo a highly abnormal and quickly abortive maturation (pseudomaturation) [ 10, 121. Finally, oocytes which have completed maturation contain a cyfostaticJactor (CSF) Differentiation 3, 3-14 (1975) - 0 by Springer-Verlag 1975
which inhibits cleavage after injection into fertilized eggs 18, 131. Whether PIF and CSF are indentical is not yet known. It has been established, however, that PIF is localized in the cortex of hormone treated oocytes and MPF in their endoplasm. Both PIF [ 101 and MPF [ 111become detectable in the cytoplasm of progesterone-treated oocytes before GV breakdown. The latter is accompanied by a marked increasedin the rate ofprotein synthesis [ 10,141.Injection of MPF into oocytes also increases protein synthesis; on the contrary, PIF is a potent inhibitor ofprotein synthesis after injection into recipient oocytes [ 101. It is well established that inhibitors of protein synthesis, such as cycloheximide, inhibit maturation, in contrast to inhibitors of DNA or RNA synthesis [ 1,4, 10, 15, 16, 17,181. However, we found that, inXenopus laevis, continuous treatment with progesterone and cycloheximide frequently induces pseudomaturation. In other experiments, we observed that normal maturation is still possible, although it is delayed, when progesterone-treated oocytes are thoroughly washed after a 5 h treatment with cycloheximide. Protein synthesis in such oocytes is still
J. Brachet et at.:
4 inhibited by 50% and it was thus concluded that full protein synthesis is not required for GV breakdom [lo]. One of the purposes of the present paper was to analyze in more detail the requirements for protein synthesis in progesterone-stimulated Xenopus oocytes: the effects of apre-treatment with concentrated cycloheximide (50 uglml) have been compared with those of simultuneous treatment with progesterone-cycloheximidemixtures and with various post-treutments [ 1 h “pulses” of cycloheximide(50 pg/ml) given 1 h, 2 h, 3 h, 4 h or 5 h after hormonal stimulation]. Thesecondpurposeofthispaper wasto fill agapinour knowledge ofthe biochemical events which follow progesterone stimulation in X . Zuevisoocytes:nothing is known so far about the energy requirements for maturation. We have thus followed oxygen consumption in progesterone-treated oocytes and studied the effects of two classical inhibitors of energy production: cyanide (KCN) and dinitrophenol (DNP).
Material and Methods All the experiments were performed on large (St. 6) oocytes ofX. laevis. The techniques used for dissecting the oocytes out of their follicles and treating them with progesterone (10 kg/ml, for various lengths of time between 2 min and 4 h) have been previously described [lo]. The inhibitors were dissolved in amphibian Ringer, unless stated otherwise, and the resultant solutions adjusted to p H 7.2-7.5 if necessary. K C N was always used at 1 mM concentration, which produces a 90% inhibition of oxygen consumption in frog eggs [ 191. Alarger range of concentrations (1 to 0.01 mM) was used in the case of DNP. A relatively high concentration (50 pg/ml) of cycloheximide was used, for 1 h pulses, since preliminary experiments have shown that protein synthesis is 90% inhibited after 1 h treatment ofXenopus oocytes with cycloheximide at that concentration. Fig. 1 describes, in a schematic way, how the pulse experiments with the 3 inhibitors were carried out. (Fig 1). The progress of maturation in the treated oocytes was followed in three different ways: inspection of the animal pole with a dissecting I
R
P
P+ I
R
P I
P
,
P P
P
R
I :
:
R
I
R
I
R
R
I
R
R
I
:
1
’
2
Results 1. Oxygen Consumption. Four independent experiments were performed and gave identical results. Fig. 2 describes a typical experiment: oxygen consumption remains the same in control and progesterone-treated oocytes during 3 to 5 h. At that time, which corresponds approximately to GV breakdown in the hormone-stimulated oocytes, oxygen consumption increases faster in the progesterone-treated oocytes than in the controls. Subsequent respiration of oocytes which have undergone maturation continues to increase faster than that of controls. However, the points shown in the second part of the curve are of doubtful significance: after being shaken for 10 h or more in the pIO2 /I00 oocytes
30 ..
20
‘7
:
R
‘
microscope and scoring the white “maturation spots”; dissection of oocytes after fixation by boiling or immersion in 5-10% TCA,in order to see the presence or absence of the GV; microscopal observation of histological sections offixed oocytes. As in our previous work [ 10, 171, groups of 2 to 4 oocytes were fixed, at various time intervals, with Zenker acetic: they were embedded in paraffin, sectioned (10 pm) and stained alternatively with Unna and Feulgen. Oxygen consumption was measured by the classical manometric method of Warburg, using Gilford microrespirometers equipped for continuous registration of the pressure changes. 150 or 200 oocytes (controls or progesterone-treated) were placed in 4 ml of Barth’s [201 mediumin each microrespirometerflask. CO, was absorbedwith0.2 ml of 1 N NaOH in the central well. In a few experiments. binding of 3H progesterone to the oocytes and protein synthesis were measured after cycloheximide treatment; the methods used have already been described [ 5 , 101.
:
R
.
;I3
Fig. 1. Schematic diagram of the pulse experiments with KCN, DNP and cycloheximide. I . Pretreatment; 2. Simultaneous treatment; 3. Post-treatments: I : inhibitor, P:progesterone, R : Ringer
10
I-
--
Y . : : : :t 3 5 7 9 13 17 I
,
t
21 (h) incubation
Fig. 2. Oxygen consumption during maturation 0-0oocytes incuboocytes incubated in 4 ml of ated in 4 ml of Barth’s medium; Barth’s medium containing 10 uglml of progesterone (mean of 3 experiments)
5
Studies on Maturation in Xenopus laeuis Oocytes
compared to the controls. Table 1 also shows that if the oocytes are washed and transfered to normal medium after a 1 h treatment with a progesterone KCN mixture, efficient reversal of inhibition occurs. No major cytological abnormality was observed in this experiment. Two pulse experiments (K, and K3) gave similar results: pretreatment during 1 h with KCN, or simultaneous treatment with KCN and progesterone during 1 h hardly affected the time course of maturation. However, in one of the experiments, a 2-h pretreatment with KCN delayed maturation for a couple of hours; this delay was followed by an “explosive” maturation: polar bodies were expulsed sooner in the oocytes which had been pretreated with KCN than in the progesterone controls. Post-treatments markedly delayed GV breakdown, in both experiments, provided that the 1 h-pulses had been applied 1 h or more after progesterone. This delay was again followed by an acceleration of maturation in one of the 2 experiments: after 8 h, 10 of the treated oocytes (out of 14) had eliminated their first polar body, while none of the progesterone controls had expulsed it. In the last (KJ experiment, a delay was still noticeable after 10 h: G V breakdown was only beginning by that time (Table 2). This difference between the two experiments is probably due to the quality of eggs rather than to different experimental conditions. In summary, the 3 experiments show that continuous treatment with KCN almost completely inhibits maturation, while pretreatment has no effect; post-treatment delays but does not prevent GV breakdown, especially when the pulses are applied at the time that oxygen consumption begins to increase. In two ofthe experiments, this delay was followed by an acceleration of first polar body expulsion when the oocytes were transferred to Ringer.
Table 1. Effects of 1 mM KCN on maturation Nr. of oocytes which underwent maturation (mat.) based on cytological observations (exp. K,) ~~
Time
Prog. alone
~
+
Prog. + 1 mM
Prog.
KCN
KCN 1 h,
1 mM
Ringer 8h Yh 11 h ~~
Total
5 mat19 4 mat16 5 math3
0 mat116 0 mat113 1 mat!l4
2 mat12 2 mat12 2 mat12
1 mat143
6 mat16
-
14 mat123
manometric flasks, increasing numbers of progesterone-treated oocytes undergo cytolysis; the controls are less fragile and more resistant to shaking. Cytolysis is known to induce a transient, but very large (10 x) increase in oxygen consumption in unfertilised frog eggs [ 191. This circumstance casts doubts upon the validity of the data obtained in long experiments. Despite this uncertainty, the fact remains that oxygen consumption increases at a time close to GV breakdown; this increase coincides, approximately, with the wellknown increase in protein synthesis [lo, 141. 2. Eflecfs ofKCN. These have been studied in three experiments. In a first experiment (K,) progesteronetreated oocytes were submitted to continuous treatment with 1mM KCN. As shown in Table 1, an almost complete inhibition of maturation was observed: out of 43 oocytes treated with KCN and cytologically examined, only 1 (about 2%) had undergone maturation in 11 h; at the same time, 14 control (progesterone-treated oocytes) out of 23 (6 1 %) had undergone GV breakdown. The only oocyte which underwent maturation, despite continuous treatment with KCN, was markedly delayed
Table 2. Effects of 1 mM KCN on maturation Nr. of oocytes which underwent maturation after treatment with 1 mM KCN. C: cytological observations. W:observations made on whole oocytes (% of maturation spots) (Exp.K3). Time
Treatment Progesterone controls 1 h pretreatment with KCN Simultaneous treatment - (1 h KCN + Prog.) Post treatments (1-h pulses) 0 h after progesterone I h after progesterone 2 h after progesterone 3 h after progesterone 4 h after progesterone
8h
w
10 h
W
C
48% 63%
2 mat15 4 mat16
100%
100%
3 mat13 3 mat13
64%
5 mat19
64%
2 mat13
45% 28% 7% 0%
12 mad36 1 mat17 1 mat18 1 mat17 1 mat18
83% 85% 50%
2 mat13
C
-
57%
2 mat13 2 mat13 2 mat13 2 mat13
J. Brachet et al.:
6 Table 3. Effects of DNP on maturation. (Exp. D, and D,) Nr. of oocytes which underwent maturation in exp D, and D,. W: observations made on whole oocytes (maturation spots). C : cytological observations*: abnormal oocytes. Exp. D,
(w)
Time
4 h 30 (W)
5 h 30 (C)
8 h (C)
16 h
Treatment Prog. (controls)
60%
2 mat13
3 mat13
82%
3 mad3
Pretreatments 1-2 h with 1 mM DNP 1-2 h with 0.1 mM DNP
0 mat129 1 mat130
1 mat16 1 mad5
0 mat16
10 mat114 12 mat112
4 mat16
0 mat1 15 2 mat115 1 mat116 6 mat126 -
0 mat12 2 mad2 1 mat13 2 mat14 3 mat13
2 mat13 2 mat14 2 mat12 4 mat/5* 1 mat/2*
Time
8 h (C)
21 h (C)
21 h (W)
Prog. (controls)
3 mat13
3 mat13
84%
Pretreatment 1 h with 0.1 mM DNP
2 mat13
3 mat13
100%
Simultaneous treatment during 30 min with prog. and DNP 0.01 mM 3 mat13
3 mat13
100%
Post-treatments with 0.1 mM DNP (1-h pulses) 0 h after progesterone 1 h after progesterone 2 h after progesterone 3 h after progesterone 4 h after progesterone
3 mat13 2 mat12 3 mat13 2 mat/3* 3 mat/3*
Post-treatments with 1 mM DNP (1-h pulses) 1 h after progesterone 2 h after progesterone 3 h after progesterone 4 h after progesterone 5 h after progesterone
0 mat13 1 mat13 1 mat12 2 mat13 2 mat12
2 mat13
3. Efects ofDNP. These were studied in three experiments, using a range of concentrations varying between 1 mM and 0.01 mM. In the first experiment (Dl), treatment with DNP was continuous. A number of errors were made in scoring the maturation spots in living oocytes and the presence of intact GV's by dissection of fixed oocytes: cytological analysis showed that, in this experiment, pseudornaturations were fairly frequent. These pseudomaturations vere similar to those previously described in oocytes treated with progesterone and DMB rifampicin [21l: a collapsed and highly basophilic GV is surrounded by a large area
3 mat17 8 mat18 5 mat17 6 mat/l4* Dead
16 h (C)
6 mat16
3 3 3 3 3
mat13 mat13 mat14 mat/4* mat/3*
72% 55% 55% 59%* 60%*
where the yolk platelets have undergone vitellolysis. Continuous treatment with 1mM or 0.1 mM DNP completely inhibited GV breakdown. Inhibition of GV breakdown was only partial with 0.0 1 M DNP: after 12 h, 57% of the continuously treated oocytes had undergone maturation against 72% ofthe progesterone controls. Cytological examination confirmed that GV breakdown is delayed in oocytes treated continuously for 12 h with 0.01 mM DNP; after 20 h, maturation was often abnormal: fuzziness or absence of the spindle prevented normal migration of the chromosomes towards the animal pole and led to abortive maturation.
Studies on Maturation in Xenopus laevis Oocytes
The 2 other experiments were 1-h “pulses” with 0.1 mM and 0.0 1 mM DNP; the results were basically similar, but not quite identical. In one of them (D,) 0.0 1 mM DNP had no effect, when applied as a post-treatment; therefore only the results obtained for pretreatments are given in Table 3. In the other experiment (D3),0.01 M DNP was dissolved in Wallace’s medium 0 [221 instead of Ringer; the main difference between the two solutions is that medium 0 contains Tris and is thus better buffered than Ringer. The results ofthe 2 experiments are summarised in Table 3.
7
As one can see from Table 3, all kinds of early treatments (pretreatment, simultaneous treatment and 1-h pulses during the 3 h which follow progesterone stimulation) slow down GV breakdown; but they never inhibit maturation irreversibly. Late treatments, applied around the time of GV breakdown, lead to abnormal maturation or even cytolysis. Cytological analysis of the treated oocytes has shown that maturation, after early treatments with DNP, can reach normal completion despite the initial delay: polar bodies had been expulsed in several of the treated
Fig. 3. Progesterone treatment during 5 h, 1 mM DNP during 1 h. Poorly differentiated spindle and pycnotic fusion of the chromosomes. Feulgen ( x 1000)
Fig. 4. Same as Fig. 3, but transfer to normal medium for 2 h30 after DNP treatment. Persistance ofnucleoli 8 h30 after hormonal stimulation. Unna (x 1000)
8
oocytes 16 or 20 h after the beginning of the experiment. More interesting, from the cytological viewpoint, are the effects of fatepulses (3-5 h after progesterone treatment). 5 h after the beginning of the experiment, there is a delay in the formation of the spindle (Fig. 3) and the chromosomes tend to fuse together. After 8 h, pigmentation becomes abnormal: the melanosomes penetrate deeply towards the interior of the oocyte. The maturation spindle is generally absent and the chromosomes are dispersed in aclear area, located at the initial position of the
J. Brachet et al.:
GV. In a few cases, several nucleoli are still present in this region (Fig. 4); others have disintegrated and only their Feulgen positive cores can be seen. The chromosomes undergo either a strong, pre-pycnotic condensation or, on the contrary, swell and form an interphase nucleus reminiscent of the female pronucleus in unfertilised eggs as in Fig. 7. Maturation is always abortive in oocytes which have been submitted to 1 h pulses with 1 mM DNP 3 to 5 h after progesterone stimulation: after 16 to 21 h, there is a slow evolution towards cytolysis and, in a few eggs, the late appearancc of a maturation spindle which usually remains
Fig. 5. Treatment: progesterone 1 h: Ringer 2 h, cycloheximide (50 pg/ml) 1 h. Ringer 16 h. Spontaneous activation: 3 nuclei stain very strongly with Feulgen (x 1000)
Fig. 6. Progesterone 1 h, cycloheximide (50 pg/ml) 1 h, Ringer 14 h. Persistance of nucleoli and remnants of the nuclear membrane 16 h after hormonal stimulation. Unna (x 1000)
Studies on Maturation in Xenopus laevis Oocytes
9
stuck in the centre of the oocyte; in very few oocytes, a poorly organised spindle, carrying pycnotic chromosomes can be found in the egg cortex. The abnormal distribution of the pigment remains unchanged until cytolysis. In summary, DNP (1 mM and 0.1 mM), like 1 mM KCN, inhibits maturation in continuous treatment; but there is a difference between the two inhibitors in pulse experiments: early treatments are much more inhibitory with DNP than with KCN; late pulses with DNP, which have no marked effects on the KCN-treated oocytes, produce detrimental effects and the oocytes become cytologically very abnormal. 4 . Effects of Cycloheximide. Our previous experiments 141 have shown that the inhibitory effects of cycloheximide are reversible only under certain conditions: single oocytes must be dissected out of the ovary before treatment and very thoroughly washed (30 min at least, with 3 successive changes inlargevolumes ofRinger) after the cycloheximide treatment in order to obtain reversal. Under these conditions, maturation is still possible in oocytes in which protein synthesis is inhibited by 50%. The purpose of the present experiments was to confirm these preliminary results and to find out whether a given period of time (before, during or after progesterone stimulation) is particularly sensitive to cycloheximide. 6 experiments have been performed; in 5 of them, cycloheximide (50 pg/ml) pulses of 2 min, 5 min, and 1 h were applied to the oocytes before, during or after hormonal treatment. In 2 experiments (C, and CJ, the length of the pulses with cycloheximide was 1 h. No inhibition of maturation was observed after pretreatment of the oocytes with the inhibitor, in contrast to our preliminary results [41suggest-
ing that binding of progesterone to its receptors might require protein synthesis. In fact, binding of 3H-progesterone to the oocytes was measured in these 2 experiments: no significant difference between cycloheximide-pretreated oocytes and the controls was found. Continuous treatment with progesterone and cycloheximide invariably led, in confirmation of our previous results, to pseudomaturation. 1 h-pulses with cycloheximide immediately following progesterone treatment, or applied 1 h or 2 h after it, delayed, but did not prevent GV breakdown: maturation spindles (1st meiotic division) were found in the cortex of the oocytes. A curious cytological observation deserves mention: 2 oocytes which had been submitted to a 1 h cycloheximide pulse 2 h after progesterone stimulation, underwent later spontaneous activation: large asters formed around several interphase nuclei which stained strongly with Feulgen (Fig. 5); aclear diastemaseparated these asters as ifthe egg had attempted cleavage. The strong Feulgen staining ofthe nuclei demonstrates that DNA synthesis had taken place in these eggs. In a 3rd experiment (CJ, on the contrary, pretreatment with cycloheximide completely inhibited maturation: as shown in Table 4, the effect of 1 h-pulses decreased in post-treatments as time after progesterone treatment went on; more and more oocytes underwent maturation, but their pigmentation became abnormal and they finally cytolysed. When the cycloheximide pulse shortly followed progesterone treatment, maturation was strongly delayed, but finally occurred in a few oocytes. Cytological analysis confirmed that maturation was greatly delayed when the cycloheximide pulse took place
Table 4. Effects of cycloheximide on maturation (Exp. C,)
Nr. of oocytes which underwent maturation in exp. C,. W: observations made on whole oocytes (maturation spots). C: cytological observations*: abnormal oocytes Time
4 h 3 0 (W)
5 h 3 0 (C)
8 h (C)
16 h (W)
16 h (C)
Treatmefit Prog.
60%
2 mad3
3 mat13
82%
3 mat13
113 1
016
016
0114
014
013 212 2.13 313
013 212 111 111
111 4/9* 118 (7 dead) Dead
213 2/3* 3/3* 2/3*
Pretreatments 1-2 h with 50 pg/ml cyclo.
Post-treatments (I-h pulses with 50 pg/ml cyclo.) 1 h after progesterone 0117 2 h after progesterone 2/18 3 h after progesterone 11/16 4 h after progesterone 12/18
10
J. Rrachet et al.:
Fig. 7. Progesterone 4 h, cycloheximide (50 pg/ml) 1 h, a n g e r 3 h30,
Abnormal pigmentation and swelling,after fusion, of the chromosomes in a large interphase nucleus Unna ( x 400)
1 h after progesterone: as shown in Fig. 6, there was no maturation spindle and a few nucleoli were still present 16 h after the beginning of the experiment. The abnormally pigmented oocytes were very similar to those described above after late pulses with DNP: the pigment has spread towards the centre of the oocyte and large interphase nuclei, similar to pronuclei, could be found: Fig. 7 shows such a giant interphase nucleus in an oocyte which had been submitted to a cycloheximide pulse 4 h after progesterone and fixed 3 h 30 later. In experiment C,,in addition to the usual 1-h pulses, a few oocytes were submitted to a much shorter treatment (5 min) with cycloheximide (50 pg/ml) immediately after progesterone stimulation. The results were very similar to those obtained in the preceeding experiments: cycloheximide completely inhibited maturation if added before, during or soon (1-2 h) after progesterone. Total inhibition was obtained even when cycloheximide had been added for only 5 min immediately after progesterone. Maturation became, again, insensitive to cycloheximideif the 1 h pulse occurred 3 h or more after progesterone; the GV's broke down, but spindle assembly was delayed and the chromosomes remained scattered in remnants of the nuclear sap for a couple of hours. After 11 h, the chromosomes underwent either pycnotic degeneration or strong decondensation in a pronucleus-like vesicle. Experiment C, was carried out in order to confirm the inhibition of maturation by very short cycloheximide pulses given after the progesterone treatment: OocYtes were first treated for 5 min with Progesterone, then for 2 or 5 min with cycloheximide (50 pg/ml) and finally
Table 5. Effects of very short pulses of cycloheximide on maturation (Exp C,)
Nr. of oocytes which underwent maturation in exp C, (cytological observations) : pseudomaturations Time
5h
9h
22 h
Treatment Progesterone alone
6 mat16
6 mat16
6 mat16
0 mat13
0 mat13
3 mat13
Immediate post-treatment Prog., cyclo 2 or 5 min Ringer Prog., cyclo 1 h, Ringer
0/6 013
0/6 013
313
Continuous treatment Prog. + cyclo
013
013
3913
Pretreatment Cyclo 1 h, prog. 5 min. Ringer
+ cycloh
516
11
Studies on Maturation in Xeaopus laevis Oocytes
Fig. 8. Progesterone 5 min., cycloheximide (50 pg/ml) 5 min, Ringer 22 h. Concentric basophilic lamellae in the cytoplasm. Unna (x 400)
extensively washed in Ringer. The results of the cytological observations are shown in Table 5 : the treatments with cycloheximide delayed maturation for many hours, but did not prevent it. After 22 h, all the treated oocytes showed a very abnormal distribution of pigment and basophilic material. The latter either formed concentric lamellae (Fig. 8) or cytasters. Maturation was always abortive, because the spindle did not assemble properly: there was either a fuzzy, large spindle, several small spindles, or even no spindle at all. The scattered chromosomes underwent pycnotic degeneration. Finally, in experiment C,, incorporation of I4Cphenylalanine was studied in oocytes which had been treated for 1 h with aprogesterone + cycloheximide (50 uglml) mixture and then thoroughly washed. Incorporation lasted 1 h and took place 1 h 30,4 h and 16 h after the beginning of the experiment. Maturation spots were present after 4 h in the progesterone controls, but they did not appear before 12 h in oocytes which had been treated with the progesterone + cycloheximide mixture. Oocytes which had been treated with the same mixture, but transferred into cycloheximide(50 pg/ml) instead of Ringer, underwent pseudomaturation. 15 h after the pulse with the progesterone-cycloheximidemixture, some ofthe oocytes had retained their initial aspect, while others showed a maturation spot. Measurements of protein synthesis were made on the two kinds of oocyte. The results of the biochemical experiments are shown in Fig. 9. As in our previous experiments [4,101, protein
synthesis increased and then strongly decreased in oocytes treated with progesterone alone. The 1 h treatment with progesterone + cycloheximide almost completely abolished protein synthesis; after extensive washing, protein
Fig. 9. Incorporation of )H-phenylalanine into proteins. Ringer ..._._ Progesterone. - .-.-.- Progesterone + cycloheximide. Each point on the graphs represents the average of 4 measurements. On the right, measurements on groups of selected oocytes: 1 : 5 normal oocytes; 2: 4 normal oocytes and 1 oocyte with maturation spot; 3 : 5 oocytes with maturation spot: 4: 2 oocytes with maturation spot and 3 oocytes with abnormal pigmentation (see text for explanations) ~
~
12 synthesis increased, first fairly rapidly, then more slowly. After 16 h, the average value for the treated oocytes was close to that found for controls maintained in Ringer. When protein synthesis was measured in the treated oocytes which showed a maturation spot after 16 h, it was found that they had incorporated much less 14Cphenylalanine than those which had not undergone maturation. We are unfortunately unable to say whether these very large difference in amino acid incorporation are due to actual protein synthesis or to reduced uptake of the labelled precursor. The results of all these experiments, taken together with the preceding ones [41 lead to the conclusion that, when a cycloheximide pulse is given shortly after progesterone, reversal of inhibition occurs after a long delay: GV breakdown took place in 5 experiments out of 7, but only after 15 h of more; later stages of maturation were always abnormal and polar-body expulsion never took place. The pulse experiments also clearly show that a marked change occurs between 2 and 4 h after progesterone stimulation (according to the female used for the experiment): while early pulses considerably delay GV breakdown, late pulses (3-4 h after progesterone) allow GV breakdown, but lead to abnormal pigmentation, decondensation or .pycnosis of the chromosomes and ultimately cytolysis.
Discussion
I . Oxygen Consumption. Since 0,consumption has never, so far as we know, been studied during maturation of amphibian oocytes, it is impossible to compare our results on Xenopus with those ofothers. We have always observed an increase in the respiratory rate at the time of GV breakdown, but it is impossible to say, at the present time, whether the two events are causally linked. Comparison with starfish oocytes does not help in drawing a conclusion. Studying 1-methyl-adenineinduced maturation in the same species (Patiria), two different authors came to opposite conclusions: oxygen consumption increases after I-methyladenine addition, but there is disagreement about the timing of this increase (at the time of GV breakdown [231 or much later, at the very end of meiosis [241). The situation prevailing in loach oocytes seems to be different from that in Xenopus: a decrease in 0, consumption, without changes in glycolysis, has been observed during maturation 1251. 2. Effects of KCN and DNP. Continuous treatment of progesterone-stimulated Xenopus oocytes with either KCN or DNP completely inhibits maturation: continuous energy production is thus required for GV breakdown. A
J. Brachet et al.:
similar conclusion has been drawn from experiments where mouse oocytes have been treated with KCN and uncouplers of oxidative phosphorylations [261. As in Xenopus, inhibition of maturation by 1 mM KCN is easily reversed by washing the mouse eggs. The fact that pretreatments with KCN and DNP do not inhibit GV breakdown speaks against the idea that energy might be required for the binding of progesterone to the plasma membrane and to the specific receptor present in the melanosomes. In pulseexperiments, post-treatments withKCN seem to be more efficient in delaying GV breakdown when they are applied 2 to 4 h after progesterone stimulation; this is the time where 0, consumption begins to increase and where GV breakdown is imminent. This delay was followed, in two of the experiments, by an acceleration of meiosis when the oocytes were transferred to normal medium. The reasons for this acceleration remain a matter of conjecture: one could imagine, for instance, that KCN inhibits the production or the activity of some factor (the cytostatic factor [8, 131 for instance) which slows down cell division; another possibility is that KCN activates some enzyme, possibly a protease, which would exert a positive control on meiotic division. The effects of post-treatments with DNP are somewhat different from those with KCN, but they are strikingly similar to those of cycloheximide: early treatments are much more effectivein inhibiting GV breakdown than late treatments; the latter produce, in contrast to KCN, detrimental effects on the structural organisation (pigment distribution, spindle formation, condensation or decondensation ofthe chromosomes) of the treated oocytes. The similarity between the effects of DNP and cycloheximideis not surprising, since in vivo and in vitro protein synthesis requires energy 1271. 3. Eflects of Cycloheximide. The present experiments confirm that, in Xenopus, continuous treatment of progesterone-treated oocytes with cycloheximide completely inhibits maturation and often leads to pseudomaturation [4, 101. On the other hand, they showthat, in contrast to what we thought previously 141, pretreatments with cycloheximide seldom inhibit maturation: binding of progesterone to the plasma membrane and the melanosome receptor does not require protein synthesis; this binding is perfectly normal under conditions where protein synthesis is 90% inhibited. The efficiency of 1 h pulses in inhibiting GV breakdown decreases as the time after hormonal stimulation increases. This is in agreement with results obtained on catfish oocytes, where maturation can be induced by corticosterone [281.Our results are also ingood agreement
Studies on Maturation in Xenopus hevis OOCYteS
with those of Dettlaff [ 1 5 , 291 and Schuetz [171, who studied the effects of puromycin pulses on GV breakdown in Rana temporaria and Ranapipiens: they found that GV breakdown is inhibited during the first 4-5 h after progesterone treatment; maturation becomes insensitive to puromycin a few hours before GV breakdown occurs in control oocytes. Unfortunately, these experiments were not analysed cytologically: errors due to pseudomaturation in scoring GV breakdown were thus possible, although pseudomaturation is a rarer event in Rana than in Xenopus. Furthermore, the effects of puromycin on protein synthesis were not measured in these early experiments: a change in permeability to the inhibitor, occurring 4-5 h after progesterone stimulation, cannot be ruled out. In Xenopus, the consequences of these late pulses are serious for the treated oocytes: the spreading of the pigment towards the centre of the oocyte and the abnormal distribution ofthe basophilic cytoplasm suggest deep alterations in the organisation of both the cortex and the endoplasm. Spindle assembly is often abnormal; when a complete spindle forms, it usually remains “arrested” in the centre of the oocyte. If the spindle eventually succeeds in reaching the cell surface, meiotic division and polar-body expulsion never take place. A comparable situation has recently been described in starfish oocytes, where the assembly of the spindle, after 1-methyladenine induced maturation does not require protein synthesis, while polar-body expulsion does [301. Probably as a result of the cytoplasmic changes induced by momentary suppression of protein synthesis, the chromosomes, in Xenopus, either decondense and form a large interphase nucleus or, on the contrary, undergo pycnotic degeneration. The reason why they undergo one or the other of these two evolutions (which have also been observed in actinomycin D treated oocytes [ 101 remains unknown. In two cases, parthenogenetic activation has been observed after a pulse which took place 2 h after hormonal stimulation: it might be the result ofslight injury during the dissection ofthe oocytes. The presence of powerful asters in such eggs shows that, in contradiction to a recent report [3 11,oocytes which have undergone in vitromaturation by progesterone treatment are capable of developing large asters. As already pointed out, there is a sharp break between “early” and “late” pulses: during the first 2-3 h of post-treatment, cycloheximide pulses slow down maturation; 1 h later, they no longer delay it, but induce the degenerative changes which have just been discussed. The timing of this break varies from one experiment to another
13
and is related to the speed at which the oocytes of a given female react to progesterone; but it always takes place 1or 2 h before GV breakdown. These observations can be best explained by assuming that cycloheximide pulses arrest the synthesis of the maturation promoting factor (MPF), whose appearance precedes GV breakdown by a couple of hours 1111. The delay in GV breakdown observedin early pulses is easily understandable if one accepts the idea that MPF production requires both energy production and protein synthesis: we know that protein synthesis increases, but does not return to its normal level, after pulsing; this partial inhibition of protein synthesis would account for a slower production of MPF and delayed GV breakdown if MPF synthesis requires protein synthesis. The situation is obviously different in the case of PIF, since pseudomaturation is linked to an inhibition of protein synthesis [4,10]; it is likely that PIF, in contrast to MPF, pre-exists in ovarian oocytes in an inactive form and that progesterone stimulates its release. This conclusion is supported by the fact that injection of melanosomes into oocytes is not followed by modifications of their GV’s; on the contrary, injection of melanosomes which have previously been equilibrated in vitro with progesterone induces pseudomaturation in the recipient oocytes [61. Finally, our results agree with the view, expressed by Smith and Ecker [ 11, that the increase in protein synthesis which follows maturation is not required for maturation itself, but might be important for later events in development: our pulse experiments show that proteins synthesised at the time of GV breakdown are less important for maturation than those synthesised earlier. Among these important “early” proteins might be the machinery for MPF synthesis. Injection ofcytoplasm taken from oocytes which have been submitted to cycloheximide pulses into recipient oocytes might provide an answer to this question.
Note Added in ProoJ Such experiments have recently been performed by K. C. Drury and S. Schorderet-Slatkine (Ce114,269,1975) and by W. J. Wasserman and Y. Masui (Exptl. Cell Res. 91,381, 1975). They lead to the conclusion that,in agreement withour own hypothesis, MPF production requires protein synthesis, while GV breakdown by MPF does not.
Acknowledgments. We wish to thank Prof. A. Ficq for help in the preparation of the microphotographs and Dr. C. Evans for improving the English,
Dedicated to Professor Etienne Wolff on the occasion of his retirement.
14
References 1. Smith, L. D., Ecker, R. E.: Curr. Topics Dev. Biol. 5, 1, 1970 2. Schuetz. A: Oogenesis. J. Biggers, A. Schuetz (eds.) p. 479, 1972 3. Schuetz, A,: Biol. of Reprod. 10, 150, 1974 4. Brachet, J., Baltus, E., Hanocq, F., Hanocq-Quertier,J., Hubert, E., Iacobelli, S., Steinert, G.: Mol. Cell. Bioch. 3, 189, 1974 5. Ozon, R., Bell&,E.: Bioch. biophys. Actu 320, 588, 1973 6. Iacobelli, S., Hanocq, J., Baltus, E., Brachet, J.: Diflerentiufion 2, 129. 1974 7. Smith. L D., Ecker, R. E.: Dev. Biol. 25, 233, 1971 8. Masui. Y . . Markert, C. I,.: J. exp. 2001.177, 129, 1972 9. Schorderet Slatkine, S.: Cell Dflerentzution 1, 179, 1972 10. Baltus. E.. Brachet, J., Hanocq-Quertier,J.. Hubert, E.: Dgerentiation 1, 127, 1973 11. Reynhout, J. K., Smith, L. D.: Dev. Biol. 38, 394. 1974 12. Steinert, G., Baltus, E., Hanocq-Quertier, J., Brachet, J.: J. ultrustr. Res. 40, 188, 1974 13. Masui, Y.: J. exp. Zool. 187, 141, 1974 14. Ecker. R. E.. Smith, L. 13.: Dev. Biol. 18, 232, 1968 15. Dettlaff, T.A.: J. Embryol. exp. Morph. 16, 183. 1966 16. Brachet, J . : Exptl. Cell Res. 48, 233, 1967 17. Schuetz. A,: J. exp. Zool. 166, 347, 1967
J. Brachet et ul. 18. Brachet. J.. Van Gansen, P., Hanocq, F.: Dev. Biol. 21, 157,
1970 19. Brachet, J.: Arch. Biol. 45, 611, 1934 20. Barth, L. G.. Barth, L. J.: J. embryol. exp. Morph. 7, 210, 1959 21. Hanocq, F., De Schutter, A,, Hubert, E., Brachet, J.:Dzflerentiution 2, 5. 1974 22. Wallace, R., Jared, D., Dumont, J., Sega, M.: J. exp. 2001.184, 321, 1973 23. Schultz,T.W., Lambert,C.C.: Exptl.Cel1 Res.81, 163, 1973 24. Houk, M. S.: Exptl. Cell Res. 83, 200, 1974 25. Ozernyuk, N. D., Zotin, A., Yurowitzky. V. G.: W.Roux’Arch. En~wicklungsinech.172, 66, 1972 26. Zeilmaker, G. H.. Vermeyden, J. P., Verhamme, C. P., van Vliet, A. C.: Eur. J. Obst. and Reprod. Biol. 4, 16, 1974 27. Panchenko. L.. Stelletskaya, N. V., Syrota, T. V., Bokhon’ko, A. I., Schuppe. N. G.: Bioch. biophys. Actu 299, 103, 1973 28. Goswami, S . V., Sundararaj. B. 1.: J. exp. Zool. 85, 327, I973 29. Dettlaff, T. A., Skoblina, M. N.:Ann. EmbryoLMorphog. Suppl. 1. 133, 1969 30. Houk, M. S., Epel, 13.: Dev. Biol. 40, 298, 1974 31. Brun, R.: Exptl. Cell Res. 88, 445, 1974
Received February 197.5
The following results were obtained: 1) The oxygen consumption of progesterone-stimulated X . laevis oocytes increases at the time of germinal vesicle (GV) breakdown. 2) Continuous treatment with 1 mM KCN, 1 mM and 0.1 mM DNP completely inhibits GV breakdown. 3) Pretreatment experiments with KCN, DNP and cycloheximide show that binding of progesterone to the plasma membrane and the specific hormone receptor requires neither energy, nor protein synthesis. 4) 1 h pulses of DNP (1 mM) or cycloheximide (50 pg/ml) were applied, at various time intervals after progesterone treatment: early pulses strongly delay, but do not prevent GV breakdown; late pulses just before GV breakdown induce a number of cytological abnormalities, which ultimately lead to cytolysis. The significance of these results is discussed and the hypothesis that synthesis of the maturation producing factor (MPF) requires both energy and protein synthesis is proposed. Introduction
Meiotic maturation, in amphibian oocytes, has been the subject of several recent reviews [ l , 2, 3,41. Addition of progesterone or certain other steroid hormones to large oocytes induces breakdown of the large germinal vesicle (VG) and expulsion of the 1st polar body. Maturation can be subdivided into a number of steps: 1) Progesterone must first react with the plasma membrane, since it is inactive if injected into oocytes. 2) The hormone binds to a receptor, which is associated with pigment granules (melanosomes) [ 5 . 61. 3) It then induces the formation of several factors in the oocyte. The most important one ofthese is the maturation promoting factor (MPF): if cytoplasm taken from a progesterone-treated oocyte is injected into a normal oocyte, maturation is induced in the latter [6, 7,8,9, 10, 111. Homogenates from hormone-stimulated oocytes also contain a pseudomaturation inducing factor (PIF): if such homogenates are injected into normal oocytes, they undergo a highly abnormal and quickly abortive maturation (pseudomaturation) [ 10, 121. Finally, oocytes which have completed maturation contain a cyfostaticJactor (CSF) Differentiation 3, 3-14 (1975) - 0 by Springer-Verlag 1975
which inhibits cleavage after injection into fertilized eggs 18, 131. Whether PIF and CSF are indentical is not yet known. It has been established, however, that PIF is localized in the cortex of hormone treated oocytes and MPF in their endoplasm. Both PIF [ 101 and MPF [ 111become detectable in the cytoplasm of progesterone-treated oocytes before GV breakdown. The latter is accompanied by a marked increasedin the rate ofprotein synthesis [ 10,141.Injection of MPF into oocytes also increases protein synthesis; on the contrary, PIF is a potent inhibitor ofprotein synthesis after injection into recipient oocytes [ 101. It is well established that inhibitors of protein synthesis, such as cycloheximide, inhibit maturation, in contrast to inhibitors of DNA or RNA synthesis [ 1,4, 10, 15, 16, 17,181. However, we found that, inXenopus laevis, continuous treatment with progesterone and cycloheximide frequently induces pseudomaturation. In other experiments, we observed that normal maturation is still possible, although it is delayed, when progesterone-treated oocytes are thoroughly washed after a 5 h treatment with cycloheximide. Protein synthesis in such oocytes is still
J. Brachet et at.:
4 inhibited by 50% and it was thus concluded that full protein synthesis is not required for GV breakdom [lo]. One of the purposes of the present paper was to analyze in more detail the requirements for protein synthesis in progesterone-stimulated Xenopus oocytes: the effects of apre-treatment with concentrated cycloheximide (50 uglml) have been compared with those of simultuneous treatment with progesterone-cycloheximidemixtures and with various post-treutments [ 1 h “pulses” of cycloheximide(50 pg/ml) given 1 h, 2 h, 3 h, 4 h or 5 h after hormonal stimulation]. Thesecondpurposeofthispaper wasto fill agapinour knowledge ofthe biochemical events which follow progesterone stimulation in X . Zuevisoocytes:nothing is known so far about the energy requirements for maturation. We have thus followed oxygen consumption in progesterone-treated oocytes and studied the effects of two classical inhibitors of energy production: cyanide (KCN) and dinitrophenol (DNP).
Material and Methods All the experiments were performed on large (St. 6) oocytes ofX. laevis. The techniques used for dissecting the oocytes out of their follicles and treating them with progesterone (10 kg/ml, for various lengths of time between 2 min and 4 h) have been previously described [lo]. The inhibitors were dissolved in amphibian Ringer, unless stated otherwise, and the resultant solutions adjusted to p H 7.2-7.5 if necessary. K C N was always used at 1 mM concentration, which produces a 90% inhibition of oxygen consumption in frog eggs [ 191. Alarger range of concentrations (1 to 0.01 mM) was used in the case of DNP. A relatively high concentration (50 pg/ml) of cycloheximide was used, for 1 h pulses, since preliminary experiments have shown that protein synthesis is 90% inhibited after 1 h treatment ofXenopus oocytes with cycloheximide at that concentration. Fig. 1 describes, in a schematic way, how the pulse experiments with the 3 inhibitors were carried out. (Fig 1). The progress of maturation in the treated oocytes was followed in three different ways: inspection of the animal pole with a dissecting I
R
P
P+ I
R
P I
P
,
P P
P
R
I :
:
R
I
R
I
R
R
I
R
R
I
:
1
’
2
Results 1. Oxygen Consumption. Four independent experiments were performed and gave identical results. Fig. 2 describes a typical experiment: oxygen consumption remains the same in control and progesterone-treated oocytes during 3 to 5 h. At that time, which corresponds approximately to GV breakdown in the hormone-stimulated oocytes, oxygen consumption increases faster in the progesterone-treated oocytes than in the controls. Subsequent respiration of oocytes which have undergone maturation continues to increase faster than that of controls. However, the points shown in the second part of the curve are of doubtful significance: after being shaken for 10 h or more in the pIO2 /I00 oocytes
30 ..
20
‘7
:
R
‘
microscope and scoring the white “maturation spots”; dissection of oocytes after fixation by boiling or immersion in 5-10% TCA,in order to see the presence or absence of the GV; microscopal observation of histological sections offixed oocytes. As in our previous work [ 10, 171, groups of 2 to 4 oocytes were fixed, at various time intervals, with Zenker acetic: they were embedded in paraffin, sectioned (10 pm) and stained alternatively with Unna and Feulgen. Oxygen consumption was measured by the classical manometric method of Warburg, using Gilford microrespirometers equipped for continuous registration of the pressure changes. 150 or 200 oocytes (controls or progesterone-treated) were placed in 4 ml of Barth’s [201 mediumin each microrespirometerflask. CO, was absorbedwith0.2 ml of 1 N NaOH in the central well. In a few experiments. binding of 3H progesterone to the oocytes and protein synthesis were measured after cycloheximide treatment; the methods used have already been described [ 5 , 101.
:
R
.
;I3
Fig. 1. Schematic diagram of the pulse experiments with KCN, DNP and cycloheximide. I . Pretreatment; 2. Simultaneous treatment; 3. Post-treatments: I : inhibitor, P:progesterone, R : Ringer
10
I-
--
Y . : : : :t 3 5 7 9 13 17 I
,
t
21 (h) incubation
Fig. 2. Oxygen consumption during maturation 0-0oocytes incuboocytes incubated in 4 ml of ated in 4 ml of Barth’s medium; Barth’s medium containing 10 uglml of progesterone (mean of 3 experiments)
5
Studies on Maturation in Xenopus laeuis Oocytes
compared to the controls. Table 1 also shows that if the oocytes are washed and transfered to normal medium after a 1 h treatment with a progesterone KCN mixture, efficient reversal of inhibition occurs. No major cytological abnormality was observed in this experiment. Two pulse experiments (K, and K3) gave similar results: pretreatment during 1 h with KCN, or simultaneous treatment with KCN and progesterone during 1 h hardly affected the time course of maturation. However, in one of the experiments, a 2-h pretreatment with KCN delayed maturation for a couple of hours; this delay was followed by an “explosive” maturation: polar bodies were expulsed sooner in the oocytes which had been pretreated with KCN than in the progesterone controls. Post-treatments markedly delayed GV breakdown, in both experiments, provided that the 1 h-pulses had been applied 1 h or more after progesterone. This delay was again followed by an acceleration of maturation in one of the 2 experiments: after 8 h, 10 of the treated oocytes (out of 14) had eliminated their first polar body, while none of the progesterone controls had expulsed it. In the last (KJ experiment, a delay was still noticeable after 10 h: G V breakdown was only beginning by that time (Table 2). This difference between the two experiments is probably due to the quality of eggs rather than to different experimental conditions. In summary, the 3 experiments show that continuous treatment with KCN almost completely inhibits maturation, while pretreatment has no effect; post-treatment delays but does not prevent GV breakdown, especially when the pulses are applied at the time that oxygen consumption begins to increase. In two ofthe experiments, this delay was followed by an acceleration of first polar body expulsion when the oocytes were transferred to Ringer.
Table 1. Effects of 1 mM KCN on maturation Nr. of oocytes which underwent maturation (mat.) based on cytological observations (exp. K,) ~~
Time
Prog. alone
~
+
Prog. + 1 mM
Prog.
KCN
KCN 1 h,
1 mM
Ringer 8h Yh 11 h ~~
Total
5 mat19 4 mat16 5 math3
0 mat116 0 mat113 1 mat!l4
2 mat12 2 mat12 2 mat12
1 mat143
6 mat16
-
14 mat123
manometric flasks, increasing numbers of progesterone-treated oocytes undergo cytolysis; the controls are less fragile and more resistant to shaking. Cytolysis is known to induce a transient, but very large (10 x) increase in oxygen consumption in unfertilised frog eggs [ 191. This circumstance casts doubts upon the validity of the data obtained in long experiments. Despite this uncertainty, the fact remains that oxygen consumption increases at a time close to GV breakdown; this increase coincides, approximately, with the wellknown increase in protein synthesis [lo, 141. 2. Eflecfs ofKCN. These have been studied in three experiments. In a first experiment (K,) progesteronetreated oocytes were submitted to continuous treatment with 1mM KCN. As shown in Table 1, an almost complete inhibition of maturation was observed: out of 43 oocytes treated with KCN and cytologically examined, only 1 (about 2%) had undergone maturation in 11 h; at the same time, 14 control (progesterone-treated oocytes) out of 23 (6 1 %) had undergone GV breakdown. The only oocyte which underwent maturation, despite continuous treatment with KCN, was markedly delayed
Table 2. Effects of 1 mM KCN on maturation Nr. of oocytes which underwent maturation after treatment with 1 mM KCN. C: cytological observations. W:observations made on whole oocytes (% of maturation spots) (Exp.K3). Time
Treatment Progesterone controls 1 h pretreatment with KCN Simultaneous treatment - (1 h KCN + Prog.) Post treatments (1-h pulses) 0 h after progesterone I h after progesterone 2 h after progesterone 3 h after progesterone 4 h after progesterone
8h
w
10 h
W
C
48% 63%
2 mat15 4 mat16
100%
100%
3 mat13 3 mat13
64%
5 mat19
64%
2 mat13
45% 28% 7% 0%
12 mad36 1 mat17 1 mat18 1 mat17 1 mat18
83% 85% 50%
2 mat13
C
-
57%
2 mat13 2 mat13 2 mat13 2 mat13
J. Brachet et al.:
6 Table 3. Effects of DNP on maturation. (Exp. D, and D,) Nr. of oocytes which underwent maturation in exp D, and D,. W: observations made on whole oocytes (maturation spots). C : cytological observations*: abnormal oocytes. Exp. D,
(w)
Time
4 h 30 (W)
5 h 30 (C)
8 h (C)
16 h
Treatment Prog. (controls)
60%
2 mat13
3 mat13
82%
3 mad3
Pretreatments 1-2 h with 1 mM DNP 1-2 h with 0.1 mM DNP
0 mat129 1 mat130
1 mat16 1 mad5
0 mat16
10 mat114 12 mat112
4 mat16
0 mat1 15 2 mat115 1 mat116 6 mat126 -
0 mat12 2 mad2 1 mat13 2 mat14 3 mat13
2 mat13 2 mat14 2 mat12 4 mat/5* 1 mat/2*
Time
8 h (C)
21 h (C)
21 h (W)
Prog. (controls)
3 mat13
3 mat13
84%
Pretreatment 1 h with 0.1 mM DNP
2 mat13
3 mat13
100%
Simultaneous treatment during 30 min with prog. and DNP 0.01 mM 3 mat13
3 mat13
100%
Post-treatments with 0.1 mM DNP (1-h pulses) 0 h after progesterone 1 h after progesterone 2 h after progesterone 3 h after progesterone 4 h after progesterone
3 mat13 2 mat12 3 mat13 2 mat/3* 3 mat/3*
Post-treatments with 1 mM DNP (1-h pulses) 1 h after progesterone 2 h after progesterone 3 h after progesterone 4 h after progesterone 5 h after progesterone
0 mat13 1 mat13 1 mat12 2 mat13 2 mat12
2 mat13
3. Efects ofDNP. These were studied in three experiments, using a range of concentrations varying between 1 mM and 0.01 mM. In the first experiment (Dl), treatment with DNP was continuous. A number of errors were made in scoring the maturation spots in living oocytes and the presence of intact GV's by dissection of fixed oocytes: cytological analysis showed that, in this experiment, pseudornaturations were fairly frequent. These pseudomaturations vere similar to those previously described in oocytes treated with progesterone and DMB rifampicin [21l: a collapsed and highly basophilic GV is surrounded by a large area
3 mat17 8 mat18 5 mat17 6 mat/l4* Dead
16 h (C)
6 mat16
3 3 3 3 3
mat13 mat13 mat14 mat/4* mat/3*
72% 55% 55% 59%* 60%*
where the yolk platelets have undergone vitellolysis. Continuous treatment with 1mM or 0.1 mM DNP completely inhibited GV breakdown. Inhibition of GV breakdown was only partial with 0.0 1 M DNP: after 12 h, 57% of the continuously treated oocytes had undergone maturation against 72% ofthe progesterone controls. Cytological examination confirmed that GV breakdown is delayed in oocytes treated continuously for 12 h with 0.01 mM DNP; after 20 h, maturation was often abnormal: fuzziness or absence of the spindle prevented normal migration of the chromosomes towards the animal pole and led to abortive maturation.
Studies on Maturation in Xenopus laevis Oocytes
The 2 other experiments were 1-h “pulses” with 0.1 mM and 0.0 1 mM DNP; the results were basically similar, but not quite identical. In one of them (D,) 0.0 1 mM DNP had no effect, when applied as a post-treatment; therefore only the results obtained for pretreatments are given in Table 3. In the other experiment (D3),0.01 M DNP was dissolved in Wallace’s medium 0 [221 instead of Ringer; the main difference between the two solutions is that medium 0 contains Tris and is thus better buffered than Ringer. The results ofthe 2 experiments are summarised in Table 3.
7
As one can see from Table 3, all kinds of early treatments (pretreatment, simultaneous treatment and 1-h pulses during the 3 h which follow progesterone stimulation) slow down GV breakdown; but they never inhibit maturation irreversibly. Late treatments, applied around the time of GV breakdown, lead to abnormal maturation or even cytolysis. Cytological analysis of the treated oocytes has shown that maturation, after early treatments with DNP, can reach normal completion despite the initial delay: polar bodies had been expulsed in several of the treated
Fig. 3. Progesterone treatment during 5 h, 1 mM DNP during 1 h. Poorly differentiated spindle and pycnotic fusion of the chromosomes. Feulgen ( x 1000)
Fig. 4. Same as Fig. 3, but transfer to normal medium for 2 h30 after DNP treatment. Persistance ofnucleoli 8 h30 after hormonal stimulation. Unna (x 1000)
8
oocytes 16 or 20 h after the beginning of the experiment. More interesting, from the cytological viewpoint, are the effects of fatepulses (3-5 h after progesterone treatment). 5 h after the beginning of the experiment, there is a delay in the formation of the spindle (Fig. 3) and the chromosomes tend to fuse together. After 8 h, pigmentation becomes abnormal: the melanosomes penetrate deeply towards the interior of the oocyte. The maturation spindle is generally absent and the chromosomes are dispersed in aclear area, located at the initial position of the
J. Brachet et al.:
GV. In a few cases, several nucleoli are still present in this region (Fig. 4); others have disintegrated and only their Feulgen positive cores can be seen. The chromosomes undergo either a strong, pre-pycnotic condensation or, on the contrary, swell and form an interphase nucleus reminiscent of the female pronucleus in unfertilised eggs as in Fig. 7. Maturation is always abortive in oocytes which have been submitted to 1 h pulses with 1 mM DNP 3 to 5 h after progesterone stimulation: after 16 to 21 h, there is a slow evolution towards cytolysis and, in a few eggs, the late appearancc of a maturation spindle which usually remains
Fig. 5. Treatment: progesterone 1 h: Ringer 2 h, cycloheximide (50 pg/ml) 1 h. Ringer 16 h. Spontaneous activation: 3 nuclei stain very strongly with Feulgen (x 1000)
Fig. 6. Progesterone 1 h, cycloheximide (50 pg/ml) 1 h, Ringer 14 h. Persistance of nucleoli and remnants of the nuclear membrane 16 h after hormonal stimulation. Unna (x 1000)
Studies on Maturation in Xenopus laevis Oocytes
9
stuck in the centre of the oocyte; in very few oocytes, a poorly organised spindle, carrying pycnotic chromosomes can be found in the egg cortex. The abnormal distribution of the pigment remains unchanged until cytolysis. In summary, DNP (1 mM and 0.1 mM), like 1 mM KCN, inhibits maturation in continuous treatment; but there is a difference between the two inhibitors in pulse experiments: early treatments are much more inhibitory with DNP than with KCN; late pulses with DNP, which have no marked effects on the KCN-treated oocytes, produce detrimental effects and the oocytes become cytologically very abnormal. 4 . Effects of Cycloheximide. Our previous experiments 141 have shown that the inhibitory effects of cycloheximide are reversible only under certain conditions: single oocytes must be dissected out of the ovary before treatment and very thoroughly washed (30 min at least, with 3 successive changes inlargevolumes ofRinger) after the cycloheximide treatment in order to obtain reversal. Under these conditions, maturation is still possible in oocytes in which protein synthesis is inhibited by 50%. The purpose of the present experiments was to confirm these preliminary results and to find out whether a given period of time (before, during or after progesterone stimulation) is particularly sensitive to cycloheximide. 6 experiments have been performed; in 5 of them, cycloheximide (50 pg/ml) pulses of 2 min, 5 min, and 1 h were applied to the oocytes before, during or after hormonal treatment. In 2 experiments (C, and CJ, the length of the pulses with cycloheximide was 1 h. No inhibition of maturation was observed after pretreatment of the oocytes with the inhibitor, in contrast to our preliminary results [41suggest-
ing that binding of progesterone to its receptors might require protein synthesis. In fact, binding of 3H-progesterone to the oocytes was measured in these 2 experiments: no significant difference between cycloheximide-pretreated oocytes and the controls was found. Continuous treatment with progesterone and cycloheximide invariably led, in confirmation of our previous results, to pseudomaturation. 1 h-pulses with cycloheximide immediately following progesterone treatment, or applied 1 h or 2 h after it, delayed, but did not prevent GV breakdown: maturation spindles (1st meiotic division) were found in the cortex of the oocytes. A curious cytological observation deserves mention: 2 oocytes which had been submitted to a 1 h cycloheximide pulse 2 h after progesterone stimulation, underwent later spontaneous activation: large asters formed around several interphase nuclei which stained strongly with Feulgen (Fig. 5); aclear diastemaseparated these asters as ifthe egg had attempted cleavage. The strong Feulgen staining ofthe nuclei demonstrates that DNA synthesis had taken place in these eggs. In a 3rd experiment (CJ, on the contrary, pretreatment with cycloheximide completely inhibited maturation: as shown in Table 4, the effect of 1 h-pulses decreased in post-treatments as time after progesterone treatment went on; more and more oocytes underwent maturation, but their pigmentation became abnormal and they finally cytolysed. When the cycloheximide pulse shortly followed progesterone treatment, maturation was strongly delayed, but finally occurred in a few oocytes. Cytological analysis confirmed that maturation was greatly delayed when the cycloheximide pulse took place
Table 4. Effects of cycloheximide on maturation (Exp. C,)
Nr. of oocytes which underwent maturation in exp. C,. W: observations made on whole oocytes (maturation spots). C: cytological observations*: abnormal oocytes Time
4 h 3 0 (W)
5 h 3 0 (C)
8 h (C)
16 h (W)
16 h (C)
Treatmefit Prog.
60%
2 mad3
3 mat13
82%
3 mat13
113 1
016
016
0114
014
013 212 2.13 313
013 212 111 111
111 4/9* 118 (7 dead) Dead
213 2/3* 3/3* 2/3*
Pretreatments 1-2 h with 50 pg/ml cyclo.
Post-treatments (I-h pulses with 50 pg/ml cyclo.) 1 h after progesterone 0117 2 h after progesterone 2/18 3 h after progesterone 11/16 4 h after progesterone 12/18
10
J. Rrachet et al.:
Fig. 7. Progesterone 4 h, cycloheximide (50 pg/ml) 1 h, a n g e r 3 h30,
Abnormal pigmentation and swelling,after fusion, of the chromosomes in a large interphase nucleus Unna ( x 400)
1 h after progesterone: as shown in Fig. 6, there was no maturation spindle and a few nucleoli were still present 16 h after the beginning of the experiment. The abnormally pigmented oocytes were very similar to those described above after late pulses with DNP: the pigment has spread towards the centre of the oocyte and large interphase nuclei, similar to pronuclei, could be found: Fig. 7 shows such a giant interphase nucleus in an oocyte which had been submitted to a cycloheximide pulse 4 h after progesterone and fixed 3 h 30 later. In experiment C,,in addition to the usual 1-h pulses, a few oocytes were submitted to a much shorter treatment (5 min) with cycloheximide (50 pg/ml) immediately after progesterone stimulation. The results were very similar to those obtained in the preceeding experiments: cycloheximide completely inhibited maturation if added before, during or soon (1-2 h) after progesterone. Total inhibition was obtained even when cycloheximide had been added for only 5 min immediately after progesterone. Maturation became, again, insensitive to cycloheximideif the 1 h pulse occurred 3 h or more after progesterone; the GV's broke down, but spindle assembly was delayed and the chromosomes remained scattered in remnants of the nuclear sap for a couple of hours. After 11 h, the chromosomes underwent either pycnotic degeneration or strong decondensation in a pronucleus-like vesicle. Experiment C, was carried out in order to confirm the inhibition of maturation by very short cycloheximide pulses given after the progesterone treatment: OocYtes were first treated for 5 min with Progesterone, then for 2 or 5 min with cycloheximide (50 pg/ml) and finally
Table 5. Effects of very short pulses of cycloheximide on maturation (Exp C,)
Nr. of oocytes which underwent maturation in exp C, (cytological observations) : pseudomaturations Time
5h
9h
22 h
Treatment Progesterone alone
6 mat16
6 mat16
6 mat16
0 mat13
0 mat13
3 mat13
Immediate post-treatment Prog., cyclo 2 or 5 min Ringer Prog., cyclo 1 h, Ringer
0/6 013
0/6 013
313
Continuous treatment Prog. + cyclo
013
013
3913
Pretreatment Cyclo 1 h, prog. 5 min. Ringer
+ cycloh
516
11
Studies on Maturation in Xeaopus laevis Oocytes
Fig. 8. Progesterone 5 min., cycloheximide (50 pg/ml) 5 min, Ringer 22 h. Concentric basophilic lamellae in the cytoplasm. Unna (x 400)
extensively washed in Ringer. The results of the cytological observations are shown in Table 5 : the treatments with cycloheximide delayed maturation for many hours, but did not prevent it. After 22 h, all the treated oocytes showed a very abnormal distribution of pigment and basophilic material. The latter either formed concentric lamellae (Fig. 8) or cytasters. Maturation was always abortive, because the spindle did not assemble properly: there was either a fuzzy, large spindle, several small spindles, or even no spindle at all. The scattered chromosomes underwent pycnotic degeneration. Finally, in experiment C,, incorporation of I4Cphenylalanine was studied in oocytes which had been treated for 1 h with aprogesterone + cycloheximide (50 uglml) mixture and then thoroughly washed. Incorporation lasted 1 h and took place 1 h 30,4 h and 16 h after the beginning of the experiment. Maturation spots were present after 4 h in the progesterone controls, but they did not appear before 12 h in oocytes which had been treated with the progesterone + cycloheximide mixture. Oocytes which had been treated with the same mixture, but transferred into cycloheximide(50 pg/ml) instead of Ringer, underwent pseudomaturation. 15 h after the pulse with the progesterone-cycloheximidemixture, some ofthe oocytes had retained their initial aspect, while others showed a maturation spot. Measurements of protein synthesis were made on the two kinds of oocyte. The results of the biochemical experiments are shown in Fig. 9. As in our previous experiments [4,101, protein
synthesis increased and then strongly decreased in oocytes treated with progesterone alone. The 1 h treatment with progesterone + cycloheximide almost completely abolished protein synthesis; after extensive washing, protein
Fig. 9. Incorporation of )H-phenylalanine into proteins. Ringer ..._._ Progesterone. - .-.-.- Progesterone + cycloheximide. Each point on the graphs represents the average of 4 measurements. On the right, measurements on groups of selected oocytes: 1 : 5 normal oocytes; 2: 4 normal oocytes and 1 oocyte with maturation spot; 3 : 5 oocytes with maturation spot: 4: 2 oocytes with maturation spot and 3 oocytes with abnormal pigmentation (see text for explanations) ~
~
12 synthesis increased, first fairly rapidly, then more slowly. After 16 h, the average value for the treated oocytes was close to that found for controls maintained in Ringer. When protein synthesis was measured in the treated oocytes which showed a maturation spot after 16 h, it was found that they had incorporated much less 14Cphenylalanine than those which had not undergone maturation. We are unfortunately unable to say whether these very large difference in amino acid incorporation are due to actual protein synthesis or to reduced uptake of the labelled precursor. The results of all these experiments, taken together with the preceding ones [41 lead to the conclusion that, when a cycloheximide pulse is given shortly after progesterone, reversal of inhibition occurs after a long delay: GV breakdown took place in 5 experiments out of 7, but only after 15 h of more; later stages of maturation were always abnormal and polar-body expulsion never took place. The pulse experiments also clearly show that a marked change occurs between 2 and 4 h after progesterone stimulation (according to the female used for the experiment): while early pulses considerably delay GV breakdown, late pulses (3-4 h after progesterone) allow GV breakdown, but lead to abnormal pigmentation, decondensation or .pycnosis of the chromosomes and ultimately cytolysis.
Discussion
I . Oxygen Consumption. Since 0,consumption has never, so far as we know, been studied during maturation of amphibian oocytes, it is impossible to compare our results on Xenopus with those ofothers. We have always observed an increase in the respiratory rate at the time of GV breakdown, but it is impossible to say, at the present time, whether the two events are causally linked. Comparison with starfish oocytes does not help in drawing a conclusion. Studying 1-methyl-adenineinduced maturation in the same species (Patiria), two different authors came to opposite conclusions: oxygen consumption increases after I-methyladenine addition, but there is disagreement about the timing of this increase (at the time of GV breakdown [231 or much later, at the very end of meiosis [241). The situation prevailing in loach oocytes seems to be different from that in Xenopus: a decrease in 0, consumption, without changes in glycolysis, has been observed during maturation 1251. 2. Effects of KCN and DNP. Continuous treatment of progesterone-stimulated Xenopus oocytes with either KCN or DNP completely inhibits maturation: continuous energy production is thus required for GV breakdown. A
J. Brachet et al.:
similar conclusion has been drawn from experiments where mouse oocytes have been treated with KCN and uncouplers of oxidative phosphorylations [261. As in Xenopus, inhibition of maturation by 1 mM KCN is easily reversed by washing the mouse eggs. The fact that pretreatments with KCN and DNP do not inhibit GV breakdown speaks against the idea that energy might be required for the binding of progesterone to the plasma membrane and to the specific receptor present in the melanosomes. In pulseexperiments, post-treatments withKCN seem to be more efficient in delaying GV breakdown when they are applied 2 to 4 h after progesterone stimulation; this is the time where 0, consumption begins to increase and where GV breakdown is imminent. This delay was followed, in two of the experiments, by an acceleration of meiosis when the oocytes were transferred to normal medium. The reasons for this acceleration remain a matter of conjecture: one could imagine, for instance, that KCN inhibits the production or the activity of some factor (the cytostatic factor [8, 131 for instance) which slows down cell division; another possibility is that KCN activates some enzyme, possibly a protease, which would exert a positive control on meiotic division. The effects of post-treatments with DNP are somewhat different from those with KCN, but they are strikingly similar to those of cycloheximide: early treatments are much more effectivein inhibiting GV breakdown than late treatments; the latter produce, in contrast to KCN, detrimental effects on the structural organisation (pigment distribution, spindle formation, condensation or decondensation ofthe chromosomes) of the treated oocytes. The similarity between the effects of DNP and cycloheximideis not surprising, since in vivo and in vitro protein synthesis requires energy 1271. 3. Eflects of Cycloheximide. The present experiments confirm that, in Xenopus, continuous treatment of progesterone-treated oocytes with cycloheximide completely inhibits maturation and often leads to pseudomaturation [4, 101. On the other hand, they showthat, in contrast to what we thought previously 141, pretreatments with cycloheximide seldom inhibit maturation: binding of progesterone to the plasma membrane and the melanosome receptor does not require protein synthesis; this binding is perfectly normal under conditions where protein synthesis is 90% inhibited. The efficiency of 1 h pulses in inhibiting GV breakdown decreases as the time after hormonal stimulation increases. This is in agreement with results obtained on catfish oocytes, where maturation can be induced by corticosterone [281.Our results are also ingood agreement
Studies on Maturation in Xenopus hevis OOCYteS
with those of Dettlaff [ 1 5 , 291 and Schuetz [171, who studied the effects of puromycin pulses on GV breakdown in Rana temporaria and Ranapipiens: they found that GV breakdown is inhibited during the first 4-5 h after progesterone treatment; maturation becomes insensitive to puromycin a few hours before GV breakdown occurs in control oocytes. Unfortunately, these experiments were not analysed cytologically: errors due to pseudomaturation in scoring GV breakdown were thus possible, although pseudomaturation is a rarer event in Rana than in Xenopus. Furthermore, the effects of puromycin on protein synthesis were not measured in these early experiments: a change in permeability to the inhibitor, occurring 4-5 h after progesterone stimulation, cannot be ruled out. In Xenopus, the consequences of these late pulses are serious for the treated oocytes: the spreading of the pigment towards the centre of the oocyte and the abnormal distribution ofthe basophilic cytoplasm suggest deep alterations in the organisation of both the cortex and the endoplasm. Spindle assembly is often abnormal; when a complete spindle forms, it usually remains “arrested” in the centre of the oocyte. If the spindle eventually succeeds in reaching the cell surface, meiotic division and polar-body expulsion never take place. A comparable situation has recently been described in starfish oocytes, where the assembly of the spindle, after 1-methyladenine induced maturation does not require protein synthesis, while polar-body expulsion does [301. Probably as a result of the cytoplasmic changes induced by momentary suppression of protein synthesis, the chromosomes, in Xenopus, either decondense and form a large interphase nucleus or, on the contrary, undergo pycnotic degeneration. The reason why they undergo one or the other of these two evolutions (which have also been observed in actinomycin D treated oocytes [ 101 remains unknown. In two cases, parthenogenetic activation has been observed after a pulse which took place 2 h after hormonal stimulation: it might be the result ofslight injury during the dissection ofthe oocytes. The presence of powerful asters in such eggs shows that, in contradiction to a recent report [3 11,oocytes which have undergone in vitromaturation by progesterone treatment are capable of developing large asters. As already pointed out, there is a sharp break between “early” and “late” pulses: during the first 2-3 h of post-treatment, cycloheximide pulses slow down maturation; 1 h later, they no longer delay it, but induce the degenerative changes which have just been discussed. The timing of this break varies from one experiment to another
13
and is related to the speed at which the oocytes of a given female react to progesterone; but it always takes place 1or 2 h before GV breakdown. These observations can be best explained by assuming that cycloheximide pulses arrest the synthesis of the maturation promoting factor (MPF), whose appearance precedes GV breakdown by a couple of hours 1111. The delay in GV breakdown observedin early pulses is easily understandable if one accepts the idea that MPF production requires both energy production and protein synthesis: we know that protein synthesis increases, but does not return to its normal level, after pulsing; this partial inhibition of protein synthesis would account for a slower production of MPF and delayed GV breakdown if MPF synthesis requires protein synthesis. The situation is obviously different in the case of PIF, since pseudomaturation is linked to an inhibition of protein synthesis [4,10]; it is likely that PIF, in contrast to MPF, pre-exists in ovarian oocytes in an inactive form and that progesterone stimulates its release. This conclusion is supported by the fact that injection of melanosomes into oocytes is not followed by modifications of their GV’s; on the contrary, injection of melanosomes which have previously been equilibrated in vitro with progesterone induces pseudomaturation in the recipient oocytes [61. Finally, our results agree with the view, expressed by Smith and Ecker [ 11, that the increase in protein synthesis which follows maturation is not required for maturation itself, but might be important for later events in development: our pulse experiments show that proteins synthesised at the time of GV breakdown are less important for maturation than those synthesised earlier. Among these important “early” proteins might be the machinery for MPF synthesis. Injection ofcytoplasm taken from oocytes which have been submitted to cycloheximide pulses into recipient oocytes might provide an answer to this question.
Note Added in ProoJ Such experiments have recently been performed by K. C. Drury and S. Schorderet-Slatkine (Ce114,269,1975) and by W. J. Wasserman and Y. Masui (Exptl. Cell Res. 91,381, 1975). They lead to the conclusion that,in agreement withour own hypothesis, MPF production requires protein synthesis, while GV breakdown by MPF does not.
Acknowledgments. We wish to thank Prof. A. Ficq for help in the preparation of the microphotographs and Dr. C. Evans for improving the English,
Dedicated to Professor Etienne Wolff on the occasion of his retirement.
14
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