/. Embryol. exp. Morph. 98,187-208 (1986)
Ig7
Printed in Great Britain © The Company of Biologists Limited 1986
Effect of alterations in follicular steroidogenesis on the
nuclear and cytoplasmic maturation of ovine oocytes
j . c. OSBORN
Regional TVF Unit, St Mary's Hospital, Hathersage Road, Manchester M13 OJH,
UK
R. M. MOOR AND I. M. CROSBY
AFRC Institute of Animal Physiology, Animal Research Station,
307 Huntingdon Road, Cambridge CB3 OJQ, UK
SUMMARY
The effects of inhibitors of follicular steroidogenesis on biochemical changes occurring in
oocytes maturing in vitro were studied using radiolabelling and polyacrylamide gel electrophoresis. These effects were correlated with previously investigated developmental abnormalities induced by the same inhibitors.
The most severe effects were generated by inhibition of 17ar-hydroxylase with the drug
SU10603 which resulted in a greatly increased ratio of progesterone to testosterone and
oestrogen. Such treatment halved the rate of meiotic maturation. Treated oocytes were analysed
individually on SDS-PAGE gels and quantitative analysis showed that the drug had induced
synthetic abnormalities even in those oocytes that resumed meiosis. This conclusion was
confirmed by separation of oocyte proteins on two-dimensional gels. The effects of SU were
reduced by delaying addition of the drug until 6 h after the beginning of maturation but were not
alleviated by the addition of exogenous oestrogen to the culture medium. When oocytes from
SU-treated follicles were transferred to inseminated, recipient ewes and recovered 24h later,
two-dimensional electrophoresis again revealed abnormalities in their protein synthetic patterns.
Almost total abolition of steroid secretion by aminoglutethimide (AG) had much less effect
on oocyte protein synthesis, although the proportion of oocytes maturing was reduced from
65 % to 46 %. The aromatase inhibitor, androstatriendione (AST) although eliminating follicular oestrogen secretion, had no effect on the rate of maturation and very little effect on protein
synthesis. These results correlate well with the effects of steroid inhibitors on fertilization and
early cleavage.
INTRODUCTION
The involvement of steroids in the maturation of mammalian oocytes is controversial. On the one hand, it has been proposed that steroids are not required for
the preovulatory resumption of meiotic maturation in rat oocytes (Lieberman
et al. 1976; Billig et al. 1983). By contrast, other workers have shown that steroids
are important for oocytes of many species for the completion of maturational
changes in both the nucleus and cytoplasm (e.g. Soupart, 1974; Thibault, 1977;
McGaughey, 1977). A more specific demonstration of the involvement of steroids
in sheep oocytes undergoing maturation in vitro has been obtained by selectively
Key words: ovine oocytes, steroidogenesis, nuclear maturation, cytoplasmic maturation,
follicle.
188
J. C. OSBORN, R. M. MOOR AND I. M.
CROSBY
altering the profile of steroids secreted by the preovulatory follicle using specific
inhibitors of follicular steroidogenesis (Moor, Polge & Willadsen, 1980). Their
results showed clearly that alterations to the steroid profile during oocyte maturation induced intracellular changes in the oocyte which were expressed as gross
abnormalities at fertilization. The incidence and severity of these aberrations
reflected the type of steroid environment within the follicle during maturation. In
these experiments, pronuclear development 24 h after transfer of the oocytes to
the oviducts of inseminated recipients was used as a measure of the effects of the
inhibitors. Although these developmental criteria provide an extremely sensitive
biological assessment of the lesions induced in oocytes by the treatments, their
origin is uncertain. Nevertheless, it is probable that these developmental abnormalities may either result from, or are associated with, anomalies in the normal
maturation of the oocyte.
It is well established that extensive reprogramming of protein synthesis occurs
in mammalian oocytes during maturation (Golbus & Stein, 1976; Schultz &
Wasserman, 1977; Van Blerkom & McGaughey, 1978; Moor, Osborn, Cran &
Walters, 1981). However, although many of these changes occur spontaneously
after the oocyte is released from the inhibitory influence of the follicle and
undergoes GVBD, it is clear that the precise pattern of changes associated with
the acquisition of full developmental competence requires follicle cell support
(Crosby, Osborn & Moor, 1981). The aim of the present investigation is therefore
to correlate the developmental effects on ovine oocytes of alterations in follicular
steroidogenesis during maturation with specific biochemical changes within the
oocyte.
MATERIALS AND METHODS
Preparation and culture of follicles
Ovaries were obtained from Welsh Mountain ewes injected on day 10-12 of the oestrous cycle
with 10 mg FSH-P (Armour, Omaha, Nebraska) in three injections 48, 40 and 24h before
slaughter. Intact, nonatretic follicles were dissected from the ovaries and cultured for 21 h using
techniques and media described previously (Moor & Trounson, 1977). Cultured follicles were
divided into seven treatment groups, outlined in Table 1, which varied with respect to the types
of gonadotrophins, steroids and steroid enzyme inhibitors added to the basic culture medium.
Gonadotrophins were added in the following standard combination: FSH (NIH-FSH-S12,
Sjugml"1), LH (NIH-LH-S18, 3/igml"1) and prolactin (NIH-P-S9, 0-02/xgrnP1). Steroid synthesis in follicles was blocked at one of three sites in the biosynthetic pathway (Fig. 1): (i) by
inhibiting the conversion of cholesterol to pregnenolone using the 20or-cholesterol oxidase inhibitor, aminoglutethimide (AG, Elipten) at a concentration of 10~ 2 M (Kahnt & Neher, 1966),
(ii) by inhibiting the 17ar-hydroxylase enzyme system with 7-chloro-3,4-dihydro-2(3-pyridyl)l-(2H)-naphthalenone (SU10603) at a concentration of 10" 4 M (Chart, Sheppard, Mowles &
Howie, 1962) or (iii) by inhibiting the aromatizing enzymes with l,4,6-androstatrien-3,17-dione
(AST) at a concentration of 10~ 6 M. Steroid supplementation consisted of the addition in one
treatment group of oestradiol-17/3 (1 /ig ml"1) to media containing SU10603. Inhibitors that were
used in preliminary tests but not included in the main study because of incomplete enzyme
inhibition were the aromatizing enzyme inhibitors 5ar-androstan-17j3-ol-3-one (dihydrotestosterone, A2570) and 4-hydroxyandrostene-3,17-dione.
Steroids and oocyte maturation
Cholesterol precursors
189
Cholesterol
1
22a,22-dihydroxycholesterol
20a--hydroxycholesterol
A^pregnenolone
Progesterone
17-hydroxyprogesterone
n-hydroxy-A'^-pregnenoloneDehydroepiandrosterone
AndrostenedioneTestosterone
Enzyme
1 20a-hydroxycholesterol
dehydrogenase
2 17a'-hydroxylase
3 Aromatizing enzymes
• Estradiol-17/3
Inhibitor
Aminoglutethimide (AG)
SU10603
1,4,6 androstatrien-3,17dione (AST)
Fig. 1. Diagrammatic representation of steroidogenic pathways in the follicle. The
numbers indicate the points of action of the inhibitory drugs used in this study.
Measurement of steroids in the culture media
The progesterone and total unconjugated oestrogen content of the medium after culture were
measured using methods and antisera previously described (Moor, Hay, Mclntosh & Caldwell,
1973). The radioimmunoassay for testosterone was validated by Moor (1977).
Electrophoretic analysis of oocyte proteins
Cumulus-enclosed oocytes were labelled at 37 °C for 3 h in 50 jul of incubation medium (Moor
et al. 1981) containing SOOjuCiml"1 of [35S]methionine (specific activity >1000Cimmol"1;
Amersham). After incubation, the cumulus-oocyte complexes were washed and denuded of
cumulus cells. The oocytes were then briefly washed in 10mM-Tris-HCl, pH7-4, collected
individually in a small volume of Tris buffer, lyophilized and frozen at -70°C until required for
electrophoresis. The quantity of labelled methionine incorporated into protein was measured
by TCA precipitation (Moor et al. 1981). Labelled proteins were analysed in one dimension as
described by Moor et al. (1981) or in two dimensions according to O'Farrell (1975), as described
by Osborn & Moor (1983a). Fluorography was as described by Moor et al. (1981) following the
methods of Bonner & Laskey (1974) and Laskey & Mills (1975). One-dimensional gels were
scanned using a microdensitometer and analysed statistically as described by Moor et al. (1981).
Transplantation and labelling of cultured oocytes
In one experiment, cumulus-oocyte complexes were removed from follicles cultured for
24 h with either gonadotrophins alone or gonadotrophins supplemented with SU10603 and
transferred to the oviducts of recipient ewes in oestrus (Moor & Trounson, 1977). Freshly
ejaculated semen was introduced into each uterine horn of recipients immediately before
transfer of oocytes. Eggs were recovered from the recipients 24 h after transfer and were labelled
with [35S]methionine prior to electrophoresis.
Supplements
Gonadotrophins
Inhibitor
None
None
FSH, LH, Prolactin
None
FSH, LH, Prolactin
SU10603
FSH, LH, Prolactin
SU10603
FSH, LH, Prolactin
SU01603 + oestradiol
FSH, LH, Prolactin
Aminoglutethimide
FSH, LH, Prolactin
1,4,6-androstatrien3,17-dione
None
None
0-21
6-21
0-21
0-21
0-21
Period of
inhibition (h)
61
26
No.
follicles
19
24
89
19
Steroid secretion (ng mg of tissue)
Progesterone
Testosterone
Oestrogen
22-9 ±4-2
23-5 ±5-9
0-1 ±0-1
70 ± 8-8
10-4 ±2-2
41-2 ±5-9
3-2 ±0-2
2-4 ±0-3
34-7 ±2-0
36-4 ±4-0
37-2 ±3-9
13-5 ±2-3
Not measured
3-1 ±0-2
5-0 ±0-4
1-3 ±0-2
5-9 ±0-9
534 ±80-1*
9-7 ±1-0
* AST crossreacted with testosterone antibody. Figure unreliable.
The following supplements were added in different combinations to follicles in the seven treatment groups: (a) gonadotrophins (FSH, 5jUg;
LH, 3//g; prolactin, 0-02jugml~1 medium); (b) steroid enzyme inhibitors (aminoglutethimide, 10~ 3 M; SU10603, 10~ 4 M; 1,4,6-androstatrien3,17-dione, 10~ 6 M) and (c) oestradiol at l^gml" 1 .
No hormones
Hormones only
SU
SU 6-21
SU + E 2
AG
AST
Treatment
group
Table 1. Outline of treatment groups and effects of inhibitors on steroidogenesis
O
O
p
o
Steroids and oocyte maturation
191
Morphological analysis of oocytes
After culture, oocytes were fixed for 24h in ethanol/acetic acid (3:1) before staining with
Lacmoid and examining using phase contrast microscopy.
RESULTS
Effects of enzyme inhibitors on follicular steroidogenesis
Previous studies have shown that the addition of either AG or SU10603 to
cultured follicles in vitro reduces the overall concentration of steroids in the
follicular fluid but that the two inhibitors have different effects on the relative
quantities of the major steroids secreted by the follicle (Moor et al. 1980). In the
present study, the measurement of steroid secretion into the culture medium
(Fig. 2; Table 1) confirmed the alterations in steroid metabolism and secretion
induced by the two drugs. Inhibiting the conversion of cholesterol to pregnenolone
with AG reduces the rate of secretion of all major classes of steroids synthesized
by the follicle, whereas inhibition of the 17or-hydroxylase system with SU10603
markedly depresses the synthesis of both androgens and oestrogens (Table 1) but
greatly increases the relative level of progesterone (Fig. 2). As expected, the
addition of the aromatizing inhibitor, AST, at explantation had no apparent effect
on the output of progesterone but markedly reduced oestrogen secretion and
appeared to increase dramatically androgen production. This latter finding is
100
80
•o
60
£ 40
20
No
hormones
GTN
GTN
GTN
+
GTN
+
GTN
+
AG
SU
SU
AST
0-21
0-21
6-21
0-21
+
Fig. 2. Graphical representation of the data in Table 1 showing the effects of gonadotrophins and inhibitors on the relative amounts of progesterone (black), testosterone
(white) and oestrogen (hatched) secreted by follicles.
192
J. C. OSBORN, R. M. MOOR AND I. M. CROSBY
readily explained by the high crossreactivity of AST with our testosterone antibody. The effects of delaying the addition of SU10603 for 6 h is also shown in Fig. 2
and Table 1. In this case, the deleterious effects of the inhibitor on oestrogen
secretion was considerably reduced and resulted in a more normal steroid profile.
This result is consistent with the observations of Moor (1977) and shows that
most of the oestrogen secreted by the follicle occurs during the first 6h after
gonadotrophin stimulation.
Steroid enzyme inhibitors and nuclear maturation
We have previously shown that the pattern of protein synthesis observed in
oocytes after culture is closely correlated with their meiotic status (Crosby et ah
1981; Osborn & Moor, 1983a). Thus, oocytes showing an unchanged or prematurational pattern of protein synthesis can be classified as being at the germinal
vesicle stage whereas oocytes showing a changed or postmaturational pattern have
undergone germinal vesicle breakdown (GVBD); the latter would include oocytes
at metaphase I as well as metaphase II (see Fig. 4; Crosby etal. 1981, plate 1). On
the basis of these findings, we have used the patterns of proteins observed in
individual oocytes to assess the effects of enzyme inhibitors on the proportion of
oocytes undergoing GVBD. The results of this analysis (Table 2) show that in the
absence of inhibitors the addition of gonadotrophins increased the proportion
of oocytes undergoing GVBD from 8% to 65%. By contrast, the additional
presence of SU10603 reduced the percentage maturing to 29 %. This effect was
partially overcome by delaying the addition of SU for 6 h but was not affected by
the presence of oestrogen in the medium. The second inhibitor, AG, also reduced
the number of oocytes undergoing GVBD, to 46 %, whereas AST had no effect.
That this method of assessing nuclear breakdown from patterns of protein
synthesis is a valid procedure is shown in Table 3. In this experiment, the
nuclear development of oocytes obtained from follicles cultured with either
gonadotrophins alone or in the presence of SU from different times after the onset
of culture was directly assessed from lacmoid-stained oocytes. The results show
that GVBD is severely inhibited by the presence of SU from the beginning of
culture and this effect is alleviated only if the addition of SU is delayed for 9 h.
Steroid enzyme inhibitors and protein synthesis
Previous work has shown that the incorporation of labelled methionine into
TCA-insoluble material by oocytes from cultured follicles is significantly increased
by the addition of LH or FSH to the culture medium and that this stimulatory
effect is prevented by the presence of SU but not by AG (Moor et al. 1981; Osborn
&Moor, 19836).
By contrast, quantitative analysis of the effect on protein synthesis of selectively
modifying steroid biosynthesis during oocyte maturation showed that the alterations in steroid secretion induced by both these inhibitors are associated with
substantial changes in the patterns of protein synthesis by the maturing oocyte
(Osborn & Moor, 19836). However, in all of these experiments, analyses were
Steroids and oocyte maturation
193
Table 2. The effects of steroid inhibitors on maturation as assessed by visual
examination of protein synthetic pattern
Treatment group
No. of oocytes
No hormones
Hormones only
SU
SU 6-21
SU + E 2
AG
AST
79
123
122
66
57
88
75
% showing changed pattern
7-6
65-0
28-7
40-9
35-1
46-6
77-3
Hormones were added to all but the first treatment group as described in Table 1.
Table 3. The effects of the addition ofSU10603 at various times after the beginning
of culture on nuclear maturation, assessed by lacmoid staining of whole oocytes
Treatment group
Total
GV
Promet
MI/MII (%)
Hormones only
SU0-24h
SU6-24h
SU9-24h
62
45
18
27
21
27
14
13
3
2
0
0
38(61)
16(36)
4(22)
14(52)
made using small groups of oocytes and the variability in synthetic activity in
individual oocytes within each treatment group was not taken into account. That
this variability is an important factor when quantitatively analysing changes in
protein synthesis is apparent from our findings in the previous section of this paper
(see Table 2). We have, therefore, re-examined the effects of gonadotrophins and
steroid enzyme inhibitors on protein synthesis by analysing both the levels of
incorporation of methionine and the patterns of proteins synthesized by individual
oocytes cultured and treated in the same way as the groups. In this way, we have
been able to make more valid comparisons between oocytes showing changed or
'postmaturationaP patterns of protein synthesis and have also been able to assess
the effects of both gonadotrophins and inhibitors on the synthetic properties of
oocytes that have not undergone maturation.
The effects of enzyme inhibitors on the incorporation of labelled methionine
into TCA-insoluble material in oocytes showing either changed or unchanged
patterns of protein synthesis are shown in Table 4. The results confirm that oocytes
from follicles exposed to gonadotrophins alone incorporate significantly more
methionine than those obtained from either untreated follicles or follicles exposed
to gonadotrophins and SU when added at explantation. Unexpectedly, however,
there was also a marginal effect (0-05 < P < 0 - 1 ) of AG on the level of incorporation. This differs from our previous observations (Osborn & Moor, 1983b) but is
consistent with the effects of AG on maturational changes shown in Table 4. It is
important to note that in all of these groups there is no significant difference in
the levels of incorporation between oocytes showing pre- and postmaturational
Figures are expressed as counts min" 1 oocyte" 1 ± S.E.M. (n).
N.S. not significant.
Table 4. Effects of inhibitors on incorporation of [35Sjmethionine into protein by oocytes
MI-MII (changed pattern)
Treatment group
+ Inhibitor
GV (unchanged pattern)
Control
/-test
—
—
—
No hormones
8 642 ±51 (64)
Hormones only
—
—
14 410 ±1437 (34)
—
SU
9678±1245(27)
P<0-05
16 707 ±1773 (23)
8 989 ±746 (42)
SU 6-21
10453 ±1647 (13)
13 258 ±1908 (23)
N.S.
14095 ±1169 (28)
SU + E 2
10 862 ±1906 (12)
6417 ±626 (9)
P<0-05
7 495 ±689 (24)
AG
12647 ±2230 (17)
8697 ±891 (34)
N.S.
9041 ±959 (25)
AST
N.S.
19943 ±2047 (15)
15 687 ±1696 (15)
12 443 ±1984 (7)
50
O
o
o
Z
>
50
o
z
CO
O
50
O
O
Steroids and oocyte maturation
195
patterns of protein synthesis. These results suggest that the increased incorporation of methionine observed during oocyte maturation is gonadotrophinregulated and is independent of nuclear and protein-synthetic changes. Since both
SU and, perhaps, AG inhibit this increase in methionine incorporation, it is
possible that steroids are involved in its regulation.
It is also evident that the presence throughout culture of exogenous oestrogen
does not overcome the suppressive effect of SU on methionine incorporation. On
the other hand, it was found that the inhibitory effect was reduced by delaying the
addition of the inhibitor for 6h. The addition of AST at explantation had no
inhibitory effect on methionine incorporation.
The data presented in Tables 2 and 3 show that steroid enzyme inhibitors can
have a broadly inhibitory effect on nuclear maturation and the associated changes
in protein synthesis. This conclusion was derived from visual assessment of protein
synthetic profiles on one-dimensional SDS gels such as those shown in Fig. 4. In
order to detect more subtle effects on the pattern of synthesis, fluorograms were
scanned and the bands quantified. Fourteen bands were selected for statistical
analysis of synthesis in six different treatment groups. Fig. 3 is a two-dimensional
representation of the relationships between the treatment groups. The table of
distances (Table 5) gives the distance between the centroids of each group.
The diagram shows that oocytes from follicles exposed to SU differed most from
untreated oocytes. Two groups of SU oocytes are included in the analysis: one
group that showed unchanged, prematurational-type patterns and a second comprising oocytes that appeared to have undergone the maturational changes. Both
of these groups are well separated (by 6 units) from both pre- and postmaturational controls. The AG- and AST-treated groups are also separated from the
postmaturational control group, although this is not very well represented in the
figure. The distance table shows that these groups are 4 units from the control
group and 3-5 units from one another. The oocytes used for these groups were all
visually assessed as being postmaturational and the analysis confirms that they are
much closer to the postmaturational than the prematurational controls.
The results of the canonical variate analysis of one-dimensional gels show that
there are quantitative differences between the protein profiles of oocytes cultured
in the presence of inhibitors of steroidogenesis and those of oocytes cultured with
gonadotrophins alone. Furthermore, in the case of SU, these differences occur
both in oocytes undergoing maturation and in those remaining at the dictyate
stage. However, since each of the protein bands separated on one-dimensional
gels may represent several different proteins, we have used two-dimensional gel
electrophoresis in an attempt to detect steroid-dependent changes in the synthesis
of specific proteins. The use of 2D gel electrophoresis for analysing oocyte proteins must, however, be approached with some caution. Difficulties in detecting
and correctly interpreting changes in protein synthesis arise from the technical
requirements of the system, which necessitate the pooling of large numbers of
oocytes, and the variability in synthetic activity between individual oocytes. It is
evident that the pooling of oocytes for 2D analysis is acceptable only if cellular
196
J. C. OSBORN, R. M. MOOR AND I. M. CROSBY
homogeneity can first be demonstrated. We have found that acceptable homogeneity can be obtained by first assessing the patterns of protein synthesis of individual
oocytes on ID gels and then combining those oocytes showing either pre- or
postmaturational patterns for subsequent analysis on 2D gels. The advantages of
6-+SU (unchanged)
/******^ + s u (changed)
—4_.
No hormones
-6--
Fig. 3. Canonical variate analysis of the effects of gonadotrophins and steroid inhibitors on protein synthesis by oocytes, separated by one-dimensional SDS-PAGE.
The first two canonical variates are plotted. Each small dot marks a single oocyte and
the large dots the centroids of the treatment groups.
Table 5. Generalized distances (the Mahalanobis D statistic, see Rao (1952)) between the
treatment groups shown in Fig. 3
No hormones
Hormones only
SU (unchanged)
SU (changed)
AG
AST
No
hormones
Hormones
only
SU
(unchanged)
SU
(changed)
—
9-97
—
5-96
9-95
—
9-66
5-67
7-61
—
AG
AST
10-61
4-02
11-16
6-77
—
10-52
4-03
10-32
5-76
3-45
—
Hormones were added to all but the first treatment group as described in Table 1. The distances
reflect the degree of difference between the patterns of protein synthesis.
Steroids and oocyte maturation
197
using this approach are clearly shown in Figs 4-7. The characteristic ID patterns
of synthesis associated with oocytes at the dictyate stage and those undergoing
meiotic maturation are shown in Fig. 4. Two-dimensional analyses of oocytes
showing only these pre- or postmaturational patterns are presented in Figs 5 and 6,
respectively. Not only do these profiles confirm that maturation is accompanied by
major changes in the patterns of protein synthesis, they also allow the firm
identification of stage-specific polypeptides. For example, polypeptides A and I
are only observed in dictyate oocytes whereas polypeptides 5 and 7 are characteristic of oocytes undergoing maturation. The difficulties involved in interpreting
2D patterns obtained by combining a mixture of oocytes showing both pre- and
postmaturational patterns are clearly illustrated in Fig. 7. As expected, the profile
is of an intermediate type with polypeptides characteristic of both stages being
present. It is clear, therefore, that comparisons between patterns that reflect
differing proportions of dictyate and maturing oocytes are invalid. We have used
this approach of prescreening oocytes on ID gels to analyse by 2D electrophoresis
the effects of enzyme inhibitors on the patterns of proteins synthesized by dictyate
and maturing oocytes.
Figs 8-11 illustrate the effects of SU on the 2D protein profiles of oocytes
cultured in intact follicles for 21 h. In each case, oocytes were prescreened on ID
gels and only those showing postmaturational patterns were used for 2D analysis.
The effects illustrated are, therefore, quite distinct from the major inhibitory
effect of SU on germinal vesicle breakdown. Fig. 9 shows the effects of continuous
exposure to SU throughout maturation, the arrows indicating the main differences
from the control pattern shown in Fig. 8. Careful,comparison of these patterns
suggests that the SU-treated oocytes have failed to undergo completely the
maturational changes in polypeptide synthesis. The arrowed polypeptides are
generally intermediate in intensity between the corresponding spots in treated
and untreated control oocytes. Figs 10 and 11 show, respectively, the effects of
delaying SU exposure for 6h and of attempting to negate the effects of SU by
adding exogenous oestrogen.
The results confirm those obtained from the ID and morphological studies,
namely that delaying exposure to the drug for 6 h reduces but does not abolish its
effects, and that added oestrogen has very little beneficial effect.
Previous studies have shown that the exposure of follicles to SU during maturation leads to abnormalities at fertilization; even oocytes that have progressed to
metaphase II are not fertilized successfully, failure of sperm to penetrate being
a common occurrence (Moor et al. 1980). In order to detect any relationship
between protein synthesis and these developmental abnormalities, oocytes from
SU-treated follicles were transferred to ewes, inseminated in vivo and recovered
after 24 h in utero for radiolabelling and electrophoresis. Figs 12 and 13 compare
the patterns of polypeptide synthesis in these oocytes with those of control oocytes
fertilized in the same way. Once again, oocytes were prescreened on ID gels
before pooling but it was not possible to detect differences between individual
oocytes, so all were combined for analysis. The arrows mark differences between
the two patterns.
198
J. C. OSBORN, R. M. MOOR AND I. M. CROSBY
Mr
x10"3
200
A
B
92
69
46
30
14-3
4
Intact GV
GVBD
IEF
x10"3
92
69
46
\
8
\c
30
10
14-3
6
GVBD
/
9 -
Steroids and oocyte maturation
199
IEF
x10' 3
92
69
46
SDS
30
J
14-3
5
Intact GV
IEF
92
69
46
SDS
30
14-3
7
50?oGVBD
Figs 4-7. Fluorographs of electrophoretic separations of labelled polypeptides synthesized by oocytes. Fig. 4 is a one-dimensional SDS gel of GV (A) and Mil (B)
oocytes demonstrating the distinctly different patterns of synthesis. Figs 5, 6 are 2D
separations of GV and Mil oocytes, respectively, previously screened on ID gels to
ensure homogeneity. Fig. 7 shows the 2D pattern produced when a mixture of GV and
Mil oocytes is separated.
200
J. C. OSBORN, R. M. MOOR AND I. M. CROSBY
IEF
Mr
x10"3
92
69
46
30
14-3
8
92
69
46
30
143
10
201
Steroids and oocyte maturation
IEF
Mr
xicr3
92
69
46
SDS
30
14-3
9
92
69
46
SDS
30
14-3
11
Figs 8-11. The effects of SU10603 on oocyte protein synthesis. Fig. 8 shows the
pattern of synthesis in control, untreated oocytes. SU was added to follicles in the
other groups at the onset of culture (Fig. 9), after 6 h (Fig. 10) and in conjunction with
oestrogen (Fig. 11).
J. C. OSBORN, R. M. MOOR AND I. M. CROSBY
202
IEF
•
Mr
x10"3
92
69
46
SDS
30
14-3
12
92
69
46
SDS
30
14-3
13
Figs 12, 13. Patterns of protein synthesis in 1-cell embryos 24 h after insemination
in vivo. The embryos in Fig. 12 were from normally matured oocytes whereas those in
Fig. 13 were from follicles exposed to SU during maturation.
203
Steroids and oocyte maturation
The two-dimensional patterns of polypeptide synthesis in oocytes from follicles
matured in vitro in the presence of AG and AST are shown in Figs 14 and 15. In
general, these patterns are very similar to those of control, in vitro -matured
oocytes.
IEF
x10"3
92
69
46
SDS
30
14-3
14
92
69
46
SDS
30
14-3
15
Figs 14,15. The effects of AG (Fig. 14) and AST (Fig. 15) on oocyte protein synthesis.
204
J. C. OSBORN, R. M. MOOR AND I. M. CROSBY
DISCUSSION
Work on the regulation of oocyte maturation by steroids has been largely
restricted to studying the inhibitory effects of the hormones on the progression of
meiosis in vitro. This was, perhaps, partly due to the observation that oocytes
could mature in follicles in vitro in the absence of steroidogenesis (Liebermann
et al. 1976). McGaughey (1977) reported that oestrogen could inhibit the nuclear
maturation of denuded pig oocytes and more recently it has been shown that
testosterone synergizes with dbcAMP (Rice & McGaughey, 1981; Racowsky,
1983) and cholera toxin (Schultz, Montgomery, Ward-Bailey & Eppig, 1983) in inhibiting GVBD and that oestrogen similarly potentiates the action of FSH (Eppig,
Freter, Ward-Bailey & Schultz, 1983). However, few studies have examined the
positive effects of steroids on maturation although McGaughey (1977) found that
progesterone and oestrogen together reduced the number of chromosomal abnormalities in pig oocytes. Earlier work showed that steroids could improve the
decondensation of the sperm head in human and rabbit oocytes (Soupart, 1974;
Thibault, 1977).
The work described in this report followed previous studies in this laboratory
that had shown that inhibitors of particular steroidogenic enzymes induced specific
abnormalities in oocyte maturation and fertilization (Moor et al. 1980). These
experiments were designed to investigate these effects in more detail by examining
endocrinological, morphological and biochemical changes in the oocyte and follicle, in order to identify the mechanisms susceptible to disruption by abnormal
steroid signals.
The processes chosen for particular attention were steroid secretion by the
follicle, morphological changes in the oocyte nucleus as it progresses from the
dictyate stage to metaphase II and changes in both the rate and patterns of protein synthesis by the oocyte. All of these have previously been studied in this
laboratory and are well characterized both in vivo and in vitro, making it possible
to identify readily any steroid-induced abnormalities. Three enzyme inhibitors
were selected for use, two of which, SU10603 and AG, had been used in the earlier
study; the third, AST, was chosen because it provided a means of selectively
reducing oestrogen secretion without abolishing androgen production.
The effects of these drugs on follicular steroid secretion were quantified by
analysis of steroids present in the medium after culture, rather than in follicular
fluid as previously. The results for SU and AG were largely confirmatory of the
earlier study; AG almost totally abolishing secretion and SU producing a grossly
distorted profile with progesterone accounting for 86 % of total production. When
SU addition was delayed for 6h, its effect was less marked, supporting earlier
studies that showed that most oestrogen secretion occurs in the first few hours
of maturation. As expected, AST also greatly suppressed oestrogen production
whilst having almost no effect on progesterone; unfortunately, AST crossreacted
with the testosterone antibody and made the accurate measurement of testosterone impossible.
Steroids and oocyte maturation
205
Having established that the three inhibitors had major but quite distinct effects
on the steroid environment of the oocyte, the next step was to relate these to
abnormalities of maturation and fertilization. In each case, exposure of the follicle
to the drug resulted in morphological and biochemical lesions in the maturing
oocyte, although the degree of disruption varied according to the inhibitor used.
The most marked abnormalities were induced by SU10603. The presence of this
inhibitor throughout maturation in vitro prevented GVBD and the associated
changes in protein synthesis in 67 % of oocytes.
Methionine incorporation was reduced by 40 % and protein synthetic patterns
were different from control oocytes, both in those oocytes remaining at GV and
those in which nuclear maturation had begun. It was, however, not possible to
identify any polypeptides that were specifically induced or totally inhibited by the
presence of SU. The patterns revealed by the 2D analysis were intermediate
between GV and normal, mature patterns. Recent studies in this laboratory
(Moor & Crosby, 1986) have revealed that the major maturation-associated
changes in protein synthesis occur at GVBD although further changes take place
during progression from MI to MIL It seems probable that the 'postmaturational'
SU groups were in fact composed of a mixture of MI and Mil oocytes. The
differences between the protein patterns of SU-treated and control oocytes after
transfer and fertilization might also be attributed to some SU-treated oocytes
being at MI. This hypothesis is supported by the previous finding that some
oocytes were at MI after 24 h in vivo (Moor et al. 1980). The deleterious effects of
SU could be reduced by delaying addition of the drug for 6h, thus allowing much
more oestrogen secretion by the follicle, but inhibition of GVBD was still marked.
Attempting to restore the steroid balance by adding exogenous oestrogen to the
culture medium had very little beneficial effect. This was in contrast to the earlier
observation that exogenous steroids could overcome the block at GVBD (Moor
et al. 1980), though in the earlier study, testosterone and 17ar-hydroxyprogestins
were added as well as oestrogens.
The effects of AG were significantly less severe, even though half the oocytes
remained at GV (compared with 35 % of controls) and methionine incorporation
was again inhibited by 50%. The effects on protein synthesis, as measured by
the canonical variate analysis, were less marked and there were no readily
identifiable, consistent differences on the 2D gels.
The aromatase inhibitor, AST, had the least effect of the three drugs tested.
Germinal vesicle breakdown and methionine incorporation were unaffected and
the effects on protein synthesis were similar to those of AG.
The inability to detect major differences at the 2D level, even though the
statistical analysis of the ID gels showed separated groups, emphasizes an important weakness of the 2D system, namely its failure to provide a quantitative
result. Furthermore, the separating system used in this investigation resolves
only those polypeptides with pis between 4 and 8. The use of alternative firstdimension gels would be necessary to analyse a greater range of molecules.
206
J. C. OSBORN, R. M. MOOR AND I. M. CROSBY
It is, of course, possible that some of the observations attributed in this study
to selective inhibitory actions of the drugs on specific cellular mechanisms do in
fact result from direct cytotoxic effects on the follicle and oocyte. While such
nonspecific activity cannot be totally discounted, the following comments argue
against this possibility. First, we have shown previously that the deleterious
effects of both aminoglutethimide and SU 10603 on oocyte development can be
considerably reduced by either delaying the addition of the inhibitors until the
second, synthetic phase of oocyte maturation or by supplementing the culture
medium from explantation with exogenous steroids (Moor et al. 1980). Second,
morphological studies of the cumulus, granulosa and theca of follicles treated
with these inhibitors have provided no evidence of cell damage (Hay & Moor,
unpublished observations cited in Moor et al. 1980) while two-dimensional gel
analysis of labelled cumulus polypeptides shows few differences in synthesis
caused by the addition of enzyme inhibitors (Osborn, Moor & Crosby, unpublished observations). Third, oocytes undergoing germinal vesicle breakdown
in the presence of SU undergo most of the associated changes in the patterns of
protein synthesis indicating that protein synthesis is not nonspecifically affected by
the drug, while oocytes from SU-treated follicles and cultured for 24 h in vivo
resume meiosis (see also Moor et al. 1980) and show patterns of protein synthesis
similar to those of control oocytes. We therefore conclude that the abnormalities
observed in oocytes exposed to the inhibitors used in this study are the result
of subtle changes in the oocyte caused by specific alterations in the steroid
environment pertaining during maturation rather than a nonspecific depression of
cellular metabolism.
The results of this study reveal more about the relationships between the many
events of maturation and the way in which they are controlled. Steroids appear to
influence two independent sets of maturational events. The first consists of GVBD
and the major changes in protein synthesis that are very tightly coupled to it. The
second comprises the totally separate increase in methionine incorporation.
Previous studies have demonstrated that not only are all these processes
gonadotrophin-regulated but that they are primarily under the control of LH
(Moor et al. 1981). A second group of maturational events is principally initiated
by the action of FSH. These include the expansion and mucification of the cumulus
cells and the associated decrease in intercellular communication between cumulus
and oocyte (Moor etal. 1981). Although these have not been studied in this investigation, there is less evidence for the involvement of steroids in their regulation.
For example, oocytes in which GVBD has been inhibited by SU still exhibit
the normal morphological changes in their cumulus cells (authors' unpublished
observations).
Overall, these investigations provide an insight into the basis for the previous
observations on the effects of steroid inhibitors on fertilization. As predicted,
the gross distortion of the steroid profile by SU was found to be more damaging
than the total shutdown of secretion induced by AG. The fact that addition of
exogenous oestrogen failed to alleviate the effects of SU, coupled with the ob-
Steroids and oocyte maturation
207
servation that selectively decreasing oestrogen output with AST was the least
disruptive of the treatments, suggests that it is the high level of progesterone
generated by SU during the early part of maturation, rather than the depression
of oestrogen, that is most damaging to the oocyte.
Aminoglutethimide and SU10603 were generously donated by Dr D. H. M. Burley, CIBA
Ltd, Horsham, Sussex. Purified gonadotrophins were a gift from the National Institute of
Arthritis, Diabetes and Digestive and Metabolic Diseases, Bethesda, Maryland, USA.
This work was supported by a Medical Research Council project grant to J. C. Osborn and
R. M. Moor.
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(Accepted 12 August 1986)