/. Embryol. exp. Morph. Vol. 56, pp. 319-335, 1980
Printed in Great Britain © Company of Biologists Limited 1980
319
Effect of follicular steroids on the maturation and
fertilization of mammalian oocytes
By R. M. MOOR, 1 C. POLGE 1 AND S. M. WILLADSEN 1
From the A.R.C. Institute of Animal Physiology, Cambridge
SUMMARY
Pronuclear development was used to measure the effects on ovine oocytes of altering
follicular steroidogenesis during maturation in vitro. Follicular steroid secretion was altered
using enzyme inhibitors and exogenous steroid supplementation. Abnormalities induced
during maturation were measured 24 h after transfer of oocytes to the oviducts of inseminated
hosts.
The presence throughout maturation of aminoglutethimide, an inhibitor of the conversion
of cholesterol to pregnenolone, reduced steroid secretion to 7% of that in controls and
decreased from 77% to 33% the number of normal oocytes. Abnormalities were substantially reduced by the addition of aminoglutethimide during the final 8 h of maturation
only.
The inhibition of 17a-hydroxylase enzymes with SU10603 reduced oestrogen and testosterone secretion to about 10% of control levels but had no effect on progestin secretion.
Only 13% of oocytes matured in the continual presence of SU10603 underwent normal
fertilization. The number of oocytes undergoing normal fertilization was increased to about
50% by (i) delaying the addition of SU10603 until the last 8 h of the maturation period or
(ii) adding exogenous steroids to follicles cultured with inhibitor from explantation.
It is concluded that oocytes require a specific intra-follicular steroid environment for the
completion of the full maturation process. Alterations to the steroid profile during maturation
induce changes in the oocyte which are expressed as gross abnormalities at fertilization.
INTRODUCTION
The importance of steroids in the maturation of mammalian oocytes is
controversial. It is the opinion of some workers that steroids are not involved
in maturation since steroid inhibitors do not prevent the resumption of meiosis
in follicles treated with LH in vitro (Lieberman et al. 1976; Tsafriri, Lieberman,
Ahren & Lindner, 1976). Other workers have, however, reported that steroids
are important in mammals for the completion of maturation changes in both
the nucleus and cytoplasm of the oocyte (Bae & Foote, 1975; McGaughey,
1977; Thibault, 1977; Moor & Warnes, 1978). The central role played by
progesterone and its metabolites in the regulation of oocyte maturation in
other species such as amphibia and fish is already firmly established (Masui,
1967; Smith, Ecker & Subtelny, 1968; Jalabert, 1976).
1
Authors' address: Institute of Animal Physiology, 307, Huntingdon Road, Cambridge
CB3 OJQ, U.K.
320
R. M. MOOR, C. POLGE AND S. M, WILLADSEN
This papsr provides new evidence for the involvement of steroids in the full
maturation of mammalian oocytes. Our approach has been to determine the
developmental consequences of selectively altering follicular steroidogenesis
during oocyte maturation. The necessary regulation of follicular steroidogenesis has been obtained by the direct application of steroid inhibitors and
steroid supplementation to isolated follicles in vitro. Physiological relevance
has been ensured by using methods of follicle culture which lead to full
maturation of the oocytes as judged by their subsequent development into
viable young (Moor & Trounson, 1977). Manipulation of the steroid environment induces subtle intracellular changes in oocytes, which although not
detectable by biochemical analysis, were nevertheless expressed in a measurable
form at fertilization.
MATERIALS AND METHODS
Preparation and culture of follicles
Over 500 non-atretic follicles (3-5-5-5 mm in diameter) were dissected from
the ovaries of Welsh Mountain sheep which had been injected with 1200 i.u.
pregnant mare serum gonadotrophin (PMSG) on day 8-10 of the cycle and
slaughtered 36-40 h later. Follicular fluid was obtained from 19 follicles
immediately after dissection. The other intact follicles were placed singly on
stainless-steel grids in plastic culture dishes and cultured under hyperbaric
conditions using the conditions and culture medium described by Moor &
Trounson (1977). Cultured follicles were divided into groups and exposed to
one of eight different treatments, 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; follicle-stimulating hormone (NIH-FSH-S12, 5 /^gml"1),
luteinizing hormone (NIH-LH-S18, 3/igml" 1 ) and prolactin (NIH-P-S9,
0-02 [ig ml"1). Steroid supplementation consisted of either oestradiol, 17/?
(1 /igml" 1 ) alone or in combination with the following steroids, oestradiol-17/?
(jug ml"1), testosterone (1 [ig ml"1), androstenedione (1 //g ml"1) ,17/?-hydroxyprogesterone (1 /ig ml"1) and progesterone (1 /tg ml"1). Steroid synthesis in
follicles was blocked at one of two sites in the biosynthetic pathway: (i) by
inhibiting the conversion of cholesterol to pregnenolone using the 20a-cholesterol
oxidase inhibitor, aminoglutethirnide at a concentration of 10~3 M (Kahnt &
Neher, 1966) or (ii) by inhibiting the 17a-hydroxylase enzyme system with
7-chloro- 3,4-dihydro-2(3-pyridyl)-l-(2H)-naphthalenone (SU-10603) at aconcentration of 10~4 M (Chart, .Sheppard, Mowles & Howie, 1962).
Inhibitors which were used in preliminary tests but not included in the
main study because of cell toxicity, incomplete enzyme inhibition or slow
action included the 7-dehydrocholesterol reductase inhibitor A.Y9944 (Ayerst
Laboratories), and the 3/?-hydroxysteroid dehydrogenase inhibitors Trilostane
(Sterling-Winthrop Research Institute) and Oxymethalone (Syntex Laboratories).
LH,
LH,
LH,
LH,
prolactin
prolactin
prolactin
prolactin
None
FSH, LH, prolactin
FSH, LH, prolactin
FSH, LH, prolactin
FSH,
FSH,
FSH,
FSH,
Gonadotrophins
Period of
inhibition
(h)
None
0-24
16-24
0-24
0-24
16-24
0-24
None
Inhibitor
None
Aminoglutethimide
Aminoglutethimide
Aminoglutethimide
SU10603
SU10603
SU1O6O3
None
Steroids
E217/?
None
None
E217/?, T, Ad,
17aHP, P
None
None
E217/?, T, Ad,
17aHP
E217, T, Ad,
17aHP, P
Hormonal supplementation
Steroid enzyme inhibitor
49
80
17
67
94
90
43
61
48
73
16
62
80
79
42
58
7
6
1
10
10
10
7
3
0
6
0
1
1
4
18
19
41
61
15
51
69
65
17
36
Oocytes excluded
,
'«.
N Oocytes
i
Transferred Recovered Degenerate Aspermic analysed
Number <Df oocytes
(The following supplements were added in different combinations or at different stages during maturation to follicles in
the eight different experiments: (a) gonadotrophins (FSH, 5/*g; LH, 3/tg; prolactin, 0-02/*gml- 1 medium), (b) steroids
all added a t - 1 / i g m h 1 (oestradiol, 17/9, E217/?; testosterone, T; androstenedione, Ad; 17a-hydroxyprogesterone, 17aHP;
progesterone, P), and steroid enzyme inhibitors (aminoglutethimide, 1 0 ~ 3 M ; SU10603, 10~ 4 M).)
Table 1. The number of cultured oocytes transferred to the oviducts of inseminated recipients and the number and
appearance of the oocytes 24 h after transplantation
U)
S'
rS
i
S"
i
322
R. M. MOOR, C. POLGE AND S. M. WILLADSEN
Transplantation and evaluation of cultured oocytes
After 24 h in culture, oocytes together with their associated cumulus cells
were removed from the follicles and placed in dissection medium at 37 °C
(Dulbecco's phosphate-buffered saline supplemented with bovine serum
albumin, 4 mg ml" 1 ; pyruvate, 0-36 M ; lactate 23-8 mM and glucose 5-5 mist).
Transfer of oocytes to the oviducts of sheep in oestrus was completed within
30-45 min of dissection (Moor & Trounson, 1977). The randomized design of
the experiments resulted in oocytes from two different treatments being transferred to almost every host animal. Freshly ejaculated semen showing wave-like
m otility was introduced into each uterine horn (0-01-0-02 ml containing 2-4 x 107
spermatozoa) of recipients immediately before transfer of oocytes. Eggs were
recovered from the recipients 24 h after transplantation. Eggs were prepared
as whole mounts and examined under phase-contrast microscopy for sperm
attachment before being fixed in acetic acid : ethanol (1:3, v/v), stained with
lacmoid and subjected to more detailed microscopical examination.
Measurement of steroids in the follicular fluid
Steroids were extracted from the follicular fluid with redistilled diethyl ether
(100: 1, v/v). The progesterone and total unconjugated oestrogen content of
the ether extracts were measured by radioimmunoassay using methods and
antisera described previously (Moor, Hay, Mclntosh & Caldwell, 1973). The
cross-reactivity of the progesterone antiserum estimated at 50 % displacement
of labelled progesterone, was, with the exception of pregnenolone (3-5%),
less than 1 % for all the major steroids secreted by the ovine follicle (Seamark,
Moor & Mclntosh, 1974). The cross-reactivity of the oestradiol-17/? antiserum
was less than 1 % for the C19 and C21 steroids; cross-reactivity with the C18
steroids was oestrone, 5 5 % ; oestradiol-17a, 6% and oestrone sulphate, 4 % .
Validation of the radioimmunoassay used for testosterone determination is
contained in the paper by Moor (1977). The cross-reactivity of the testosterone
antiserum with C18 and C 2i steroids was less than 0-01 %, but for the C19
steroids was 5a-dihydrotestosterone, 100%; 5/?-androstane-3a,17/?-diol,
4 3 % ; 5a-androstane-3a,17/?-diol, 4 1 % ; androst-5-en-3/?, 17^-diol, 4 % and
androstenedione, 0-01 %. The term 'testosterone' is used in this paper because
previous studies have shown that the androgens which cross-react with our
testosterone antibody account for less than 5 % of the steroid in that fraction.
The minimum amount of steroid that could be distinguished from the blank
was 10 pg for oestrogen, 15 pg for testosterone and 30 pg for progesterone. The
within-assay and between-assay coefficients of variation for the three assays
were 6-1% and 12% for progesterone, 5-9% and 7% for oestrogen and
4-7% and 8% for testosterone.
Steroid-oocyte interactions
_
323
RESULTS
Sheep oocytes, removed from follicles after a 24 h culture period in defined
conditions, become fertilized and develop into young when transferred to
inseminated recipients (Moor & Trounson, 1977). The groups of follicles outlined in Table 1 were cultured under the same conditions as the above except
that follicular steroidogenesis was selectively altered by enzyme inhibitors and
steroid supplementation. Oocytes from 501 follicles cultured in the eight
groups were transferred to inseminated recipients and 24 h later 458 (91 %)
oocytes were recovered from the oviducts of the recipients. On examination,
12% of the recovered oocytes were classified as degenerate and 11% as
aspermic. Both of these types of oocytes were excluded from further consideration and all subsequent analyses were restricted to the 355 (77%) oocytes
that had at least one spermatozoon attached to the zona pellucida or embedded
in the cytoplasm.
Oocyte development after inhibition of total steroid synthesis
It is clear from Table 2 that aminoglutethimide, added to the culture medium
at explantation sharply reduced (P < 0-001) the secretion of all the major
classes of steroids secreted by follicles. By contrast, addition of aminoglutethimide 16 h after explantation markedly depressed progesterone and
testosterone secretion below that of controls (P < 0-001) but had little effect on
the levels of oestrogen. This latter rinding is readily explained by the fact that
oestrogen secretion occurs in the early phase of maturation process while
progestin secretion is confined to the last phase.
Figure 1 summarizes the response of the oocyte, as measured by changes
during fertilization, of totally inhibiting steroid secretion during maturation
in vitro. Oocytes from follicles cultured in the absence of inhibitors (control
group, Fig. 1A) showed normal development of male and female pronuclei
in the great majority of oocytes (77%). In these normal oocytes, both the
male and female pronuclei had generally developed to the late pronuclear
stage and the mid-piece of the sperm tail was clearly visible (Fig. 2). A few
oocytes had reached the stage of syngamy (Figs 3, 4). The incidence of polyspermy in oocytes with normal development of the female pronucleus was
negligible. Thirteen oocytes (19 %) failed to complete meiosis. Nine had not
developed beyond metaphase-I and four, which had been penetrated by spermatozoa, remained at metaphase-II.
Inhibiting steroidogenesis by adding aminoglutethimide at explantation
reduced to only 33 % the proportion of oocytes with morphologically normal
pronuclear development (Fig. 1B). The most striking abnormality in this group
was the large proportion of oocytes that failed to complete meiosis. The
number of oocytes blocked at or before metaphase I amounted to 33 %, but
in a further 22% of oocytes the chromosomes remained at metaphase II
0-24
16-24
0-24
16-24
—
—
—
—
0
24
24
24
24
24
Aminoglutetbimide (10~3 M)
Aminoglutethimide CIO"3 M)
SU1O6O3 (10-4 M)
SU1O6O3 (10-4 M)
(h)
Inhibitor
Period
(h)
Period
Enzyme inhibitor
29
74
71
47
66
23
follicles
No
2485 ±186
3729 ±517
411 ±66
2686 ±161
525 ±128
7461± 895
Oestrogen
1375 ±93
1097 ±163
208 ±42
277 ±42
55 ±14
1384 ±208
Testosterone
(pmoles ml" )
1
544 ±59
4919 ±611
99 ±22
426 ±73
4487 ±540
4519 ±506
Progesterone
Table 2. Mean concentration (±S.E.M.) oj unconjugated oestrogen, testosterone and progesterone in Jollicular fluid at
explantation and after 24 h culture in medium supplemented with gonadotrophins and inhibitors of steroid synthesis
{see Table 1 Jor details)
W
1/3
O
r
i—i
>
d
O
O
p
o
o
Steroid-oocyte interactions
(A)
80
-
60
-
40
-
20
-
n
(B) Aminoglutethimkle added
at explantation
Control group
,
325
WA
1
(C) Aminoglutethimide added
16 h after explantation
(D) Aminoglutethimide + steroids
added at explantation
40 -
20 -
M I
M II
PN
MI
Mil PN
Development of oocyte nucleus
Fig. 1. Effect of blocking steroid synthesis during maturation in vitro on the
development of oocytes examined 24 h after transfer to inseminated hosts.
Illustrated are the percentage of oocytes at the germinal vesicle (GV), first and
second metaphase (MI, Mil) or pronuclear (PN) stages of development, together
with the associated extent of sperm development in oocytes at each stage of meiosis.
Sperm development is classified as: attachment but no penetration (D), monospermic penetration with abnormal decondensation (11), polyspermic penetration
(E2) and normal male pronuclear formation ( • ) .
The following supplements were added to the follicles during the 24 h maturation
period. (+) Gonadotrophin added at explantation (control group); (B) gonadotrophin and aminoglutethimide (10~3 M) added at explantation; (C) gonadotrophin at explantation and aminoglutethimide (10~3 M) added 16 h later; (D)
gonadotrophin, aminoglutethimide (10~3 M) and exogenous steroids added at
explantation.
326
R. M. MOOR, C. POLGE AND S. M. WILLADSEN
following sperm penetration (Fig. 5). The majority of these oocytes were penetrated by more than one spermatozoon and the development of the male
pronuclei was also abnormal. In some oocytes the male pronuclei were arrested
at the stage of sperm head swelling and decondensation (Fig. 6) and in others
the pronuclei appeared to be misshapen and fragmenting (Fig. 7).
Addition of aminoglutethimide 16 h after explanation (Fig. 1C) increased
the number of oocytes with normal pronuclei from 33 % (when aminoglutethimide was added at explantation) to 59 %. Female pronuclear development
occurred normally in 88 % of all oocytes. None was blocked at metaphase II
after sperm penetration, but in 12% meiosis had not progressed beyond
metaphase I.
A similar high incidence (76 %) of normal female pronuclear development
occurred in oocytes from follicles cultured from explantation with aminoglutethimide but with exogenous steroid supplementation (Fig. 1D). A further 14 %
of oocytes were at metaphase II but these had not been penetrated by
spermatozoa. There was a relatively high incidence of polyspermy (22 %) and
this together with another 11 % of the oocytes in which male pronuclear
formation was retarded (Fig. 9) resulted in only 41 % containing normal
male and female pronuclei.
Oocyte development after inhibition of 17<x-hydroxylase
As expected, inhibition of the 17a-hydroxylase system with SU10603 sharply
reduced oestrogen and androgen secretion (P < 0-001) but had no apparent
effect on the output of progesterone (see Table 2). With the exception of
oestrogen which was increased above control levels (P < 0-01), other steroid
FIGURES
2-7
Figs. 2-4. Normal sequence of nuclear events during fertilization of oocytes
matured in control follicles in vitro (see Fig. 1A). Scale bar represents 20/*M.
Fig. 2. Male and female pronucleus together with the midpiece from the fertilizing
spermatozoon.
Fig. 3. Oocyte at syngamy.
Fig. 4. Oocyte showing first cleavage division.
Figs. 5-7. Abnormalities during fertilization induced in oocytes by the inhibition
of steroid synthesis during maturation with aminoglutethimide (see Fig. 1B). Scale
bar represents 20 /IM.
Fig. 5. Oocyte nucleus blocked at metaphase II and showing the first polar body.
An abnormal male pronucleus in this polyspermic oocyte is shown in Fig. 7.
Fig. 6. Polyspermy and arrested sperm decondensation. Development of the
female nucleus in this oocyte was arrested at metaphase II.
Fig. 7. An abnormal and fragmenting male pronuclei in the oocyte depicted in
Fig. 5. A second penetrated sperm shows almost no decondensation or swelling of
the sperm head.
Steroid-oocyte interactions
/ V .
v,v*-* :/
327
328
R. M. MOOR, C. POLGE AND S. M. WILLADSEN
secretion was unaffected by the addition of SU10603 at 16 h after explantation
of follicles.
The addition of the 17a-hydroxylase inhibitor, SU10603, at explantation
resulted in an almost total disruption of normal oocyte development and
fertilization (Fig. 8 A). The abnormalities were characterized by (i) an inability
of spermatozoa to penetrate the zona pellucida after attachment to it (73 %
not penetrated) and (ii) by a block in meiosis in 57 % of oocytes at or before
the formation of the first metaphase plate (Figs 10, 11).
The deleterious effect of SU10603 was considerably reduced both by delaying
the addition of the inhibitor for 16 h (Fig. 8B) and by supplementing the
medium containing SU10603 with exogenous steroids (Fig. 8C). Oocytes from
follicles exposed to SU10603 between 16 and 24 h after explantation had
mostly developed to metaphase II or had formed pronuclei (93 %). Only 20 %
had not been penetrated by spermatozoa and the majority of the pronuclear
eggs were normal. The presence throughout culture of exogenous steroids
together with SU10603 also decreased problems of sperm penetration (only
12 % not penetrated) and increased the proportion of oocytes in which meiosis
had been completed (71 % at metaphase II or pronuclear stage). Normal male
and female pronuclei were present in 51 % of oocytes, but the majority of the
immature oocytes that had been penetrated by spermatozoa were polyspermic
(Fig. 12).
The formation of normal male and female pronuclei occurred in only 5 %
of oocytes obtained from follicles cultured in the absence of gonadotrophins
but in the presence of exogenous steroids (Fig. 8D). Meiosis was blocked at
or before metaphase I in 18% of oocytes and although 52% had reached
metaphase II at the time when they were recovered from the recipient animals,
a large proportion of these had not been penetrated by spermatozoa. A relatively
high proportion of the pronuclear eggs were also polyspermic and many of the
male pronuclei in these oocytes as well as in the immature oocytes penetrated
by spermatozoa showed irregularities in decondensation and were fragmenting
(Figs 13, 14).
DISCUSSION
The results demonstrated that alterations to the normal profile of steroids
secreted during maturation induced intracellular changes in oocytes which are
expressed clearly at fertilization. The different abnormalities that develop
during the period of interaction between the sperm and oocyte reflect the type
of steroid environment pertaining during maturation. It would seem that
imbalances in the steroid profile affect oocyte maturation more severely than
the total inhibition of steroid biosynthesis.
The addition of aminoglutethimide effectively reduced overall follicular
steroid secretion and sharply increased the incidence of abnormalities within
the oocyte. These results cannot, however, for the following reasons be inter-
Steroid-oocyte interactions
(A)
100 r
SU10603 added
at explanation
329
(B)
SU10603 added 16 h
after explain tation
(D)
Steroids only added
at explantation
80
60
40
20
(C) SU 10603 + steroids added
at explantation
40 -
20 -
Mil
PN
GV
MI
Mil
PN
Development of oocyte nucleus
Fig. 8. Effect of selectively altering the profile of steroids during maturation
in vitro on the development of oocytes examined 24 h after transfer to inseminated
hosts. Illustrated are the percentages of oocytes at the germinal vesicle (GV), first
and second metaphase (M I, M II) and pronuclear stages of development, together
with the associated extent of sperm development in oocytes at each stage of
meiosis. Sperm development is classified as: attachment but not penetration (D);
monospermic penetration with abnormal decondensation (H); polyspermic penetration ( 0 ) ; and normal male pronuclear formation ( • ) .
The following supplements were added to the follicles during the 24 h maturation
period: (A) gonadotrophin and SU1O6O3 (10~4 M) added at explantation; (B)
gonadotrophin at explantation and SU10603 (10~4 M) 16 h later; (C) gonadotrophin,
SU1O6O3 (10~4 M) and exogenous steroid added at explantation; (D) exogenous
steroid added at explantation.
330
R. M. MOOR, C. POLGE AND S. M. WILLADSEN
Steroid-oocyte interactions
331
preted as measuring the capacity of oocytes to mature in the total absence of
steroids. Firstly, it has been found that the block to follicular steroidogenesis
induced by aminoglutethimide requires a few hours to become effective (H. M.
Dott, D. Green & R. M. Moor, unpublished observations). Secondly, the high
levels of secreted steroid contained within the follicle (see Table 2) are not
altered or removed by the addition of the inhibitor. Finally, it is evident, from
the present results that aminoglutethimide is effective in disrupting oocyte
development during the first phase of the maturation process only; addition
of the inhibitor during the second phase of maturation was without effect on
subsequent pronuclear development. It is therefore probable that the period
during which the oocyte is most sensitive to steroid deprivation coincides in
aminoglutethimide treated follicles with the period of highest residual steroid
availability. Thus, the real effect on the oocyte of a total absence of steroids
may be considerably more severe than is suggested by the results of our
experiments. This possibility is supported by preliminary evidence which suggests that the addition of antisera to oestrogen and testosterone, together with
aminoglutethimide, induces more abnormalities in oocytes than the inhibitor
alone (R. M. Moor, unpublished observations).
A comparison of the effects of blocking either the 20a cholesterol or the
17a-hydroxylase system indicates that the maturing oocyte is probably more
sensitive to an imbalance rather than an overall reduction in steroid levels.
This postulate accords with the report by Soupart (1974) that human oocytes
matured in vitro require a sequence of steroid additions in order to develop
fully the potential to sustain subsequent normal male pronuclear development.
FIGURES
9-14
Abnormalities during fertilization induced in oocytes by selectively altering
steroid concentrations in follicles during maturation. Scale bar represents 20 JUM.
Fig. 9. Female pronucleus and penetrated spermatozoon with arrested development
in an oocyte cultured with, aminoglutethimide and exogenous steroid supplementation (see Fig. ID).
Fig. 10. Oocyte nucleus at the germinal vesicle stage and numerous spermatozoa
which had become embedded in the zona pellucida (removed during fixation) but
had failed to enter the cytoplasm of an oocyte matured in the presence of SU10603
(see Fig. 8 A).
Fig. 11. Oocyte nucleus arrested at metaphase I and spermatozoa attached to the
zona pellucida (removed during fixation); spermatozoa were unable to penetrate
oocytes matured in the presence of SU10602 (see Fig. 8A).
Fig. 12. Oocyte nucleus at the germinal vesicle stage with polyspermy and sperm
nuclei showing arrested swelling and decondensation in an oocyte matured with
SU1O6O3 and exogenous steroids (see Fig. 8C).
Fig. 13. Fragmentation of the male pronucleus in a oocyte cultured with
exogenous steroids but without gonadotrophic support (see Fig. 8D).
Fig. 14. Metaphase II chromosomes and first polar body together with an
abnormal pronucleus in an oocyte matured with exogenous steroids but without
gonadotrophic support (see Fig. 8D).
332
R. M. MOOR, C. POLGE AND S. M. WILLADSEN
The requirement for a strictly regulated sequence of steroid changes during
maturation may explain why exogenous steroids did not completely reverse
the action of the inhibitors. The addition of steroids together with aminoglutethimide restored female pronuclear development to control levels but
did not restore normal male pronuclear development. This incomplete reversal
could have been caused by the addition at explantation of equal amounts of
oestrogen, androgens and progestins; the resultant steroid environment clearly
differed substantially from that pertaining in vivo (Moor, 1978). By contrast
only oestrogen, androgen and 17a-hydroxylated progestins were included when
the 17a-hydroxylase inhibitor was used; the resultant more normal steroid
profile may explain the higher incidence of reversal in that experiment. Steroids
other than those supplied (the follicle secretes at least 12-16 different steroids)
or the precise concentration of added steroid may also be of significance in
oocyte maturation. With the numerous steroid variables likely to be important
in oocyte maturation it is perhaps unrealistic to expect complete reversal of
enzyme inhibition until considerably more detailed information on steroidoocyte interactions becomes available.
It is possible that some of the abnormalities observed in oocytes exposed
to the inhibitors used in this study could have resulted from direct toxic
effects of the drugs. While such direct cytotoxic effects cannot be discounted,
the following observations provide evidence against this possibility. Firstly,
oocytes exposed to both inhibitors develop entirely normally provided only
that the drugs are not added until the second phase of maturation. Secondly,
the simple addition of steroids to cultures containing one of the inhibitors
(SU10603) increases from 13 to 50 % the number of oocytes undergoing normal
development and fertilization. Thirdly, morphological studies of the cumulus,
granulosa and theca of follicles treated with these inhibitors provided no
evidence of cell damage (M. F. Hay & R. M. Moor, unpublished observations).
The precise mechanism by which steroid imbalances affect the oocyte is
unclear. It seems unlikely that maturation in mammalian oocytes is initiated
by the same kind of steroid-membrane interactions that initiate maturation
in amphibian oocytes (Godeau, Schorderet-Slatkine, Hubert & Baulieu, 1978).
A favoured alternative hypothesis for mammals suggests that the disruption
of gap junctions between cumulus cells and oocytes initiates maturation by
leducing intercellular uptake into oocytes of inhibitors which block meiosis;
the most likely inhibitor being cyclic AMP (Anderson & Albertini, 1976;
Gilula, Epstein & Beers, 1978; Dekel & Beers, 1978). Recent experiments on
the role of hormones in gap junction function suggest that steroids have a minor
but statistically significant effect on the passage of molecules from cumulus
cells to oocytes (Moor, Smith & Dawson, 1979). However, the major regulators
of intercellular coupling and amino acid transport across the oolemma, namely
FSH and LH, are not dependent upon steroid synthesis for their action on the
membrane (Moor & Smith, 1978, 1979; Moor et al. 1979). It therefore seems
Steroid-oocyte interactions
333
unlikely that the action of steroids is primarily on the membrane of the
oocyte.
The early sequence of maturational changes in the nucleus leading to germinal
vesicle breakdown and formation of the first metaphase plate are allegedly
independent of steroid support (Lieberman et al. 1976). The evidence of
McGaughey (1977) indicates, however, that the absence of steroids during
the later stages of maturation increases the incidence of chromosomal abnormality especially at telophase I and metaphase II. The present results
support the observations of McGaughey (1977) and show that major changes
in follicular steroidogenesis during maturation result in severe nuclear abberations. The addition of exogenous steroids together with the steroid inhibitor,
particularly when aminoglutethimide is used, reduce substantially the incidence
of chromosomal abnormalities in the oocytes.
Oocytes, exposed before maturation to sperm, become penetrated but lack
the cytoplasmic factors that induce decondensation and formation of the male
pronucleus (reviewed by Thibault, 1977). A specific factor responsible for the
transformation of the sperm into a male pronucleus has not been characterized
but is probably amongst those compounds whose synthesis is induced during
normal oocyte maturation (Golbus & Stein, 1976; Schultz & Wasserman,
1977; Warnes, Moor & Johnson, 1977; Van Blerkom & McGaughey, 1978).
Evidence from Soupart (1974) and Thibault, Gerard & Menezo (1975) suggests
that steroids are important for the synthesis of the so called pronucleus growth
factor (MPGF). Our results support this contention.
We have used developmental criteria in this study to detect abnormalities
during oocyte maturation because of the extreme sensitivity of such biological
tests. It has been our purpose to extend the study by correlating developmental
aberrations with specific synthetic changes in the oocyte. We have, however,
so far failed to detect clear steroid-dependent changes in protein synthesis in
oocytes using polyacrylamide slab gels containing sodium dodecyl sulphate
(R. M. Moor & G. M. Warnes, unpublished observations). The substantially
greater degree of protein separation obtained using isoelectric focusing and
SDS polyacrylamide electrophoresis (O'Farrell, 1975), may however reveal
differences in synthesis which were not resolved by one-dimensional gel electrophoresis.
The authors acknowledge with gratitude gifts of aminoglutethimide phosphate and
SU10603 from Dr D. H. M. Burley, CIBA Pharmaceutical Company (Horsham, Sussex);
oxymethalone from Dr H. C. Anderson, Syntex Corporation (Palo Alto, California),
AY-9944 from Dr D. Dvornik, Ayerst Research Laboratories (Montreal, Canada) and
Trilostane from Dr F. C. Nachod, Sterling-Wintbrop Research Institute (Renssselaer, New
York). The purified gonadotrophins used in this study were generously donated by the
National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of
Health, Bethesda, Maryland. An MMB grant to one of us (S.M.W.) is gratefully acknowledged.
334
R. M. MOOR, C. POLGE AND S. M. W I L L A D S E N
REFERENCES
ANDERSON, E. & ALBERTINI, D. F. (1976). Gap junctions between the oocyte and companion
follicle cells in the mammalian ovary. /. Cell Biol. 71, 680-686.
BAE, I. H. & FOOTE, R. H. (1975). Effects of hormones on the maturation of rabbit oocytes
recovered from follicles of various sizes. /. Reprod. Fert. 42, 357-360.
CHART, J. J., SHEPPARD, H., MOWLES, T. & HOWIE, N. (1962). Inhibitors of adrenal corticosteroid 17a-hydroxylation. Endocrinology 71, 479-486.
DEKEL, N. & BEERS, W. H. (1978). Rat oocyte maturation in vitro: Relief of cyclic AMP
inhibition by gonadotrophins. Proc. natn. Acad. Sci., U.S.A. 75, 4369-4373.
GILULA, N. B., EPSTEIN, M. L. & BEERS, W. H. (1978). Cell-to-cell communication and
ovulation. J. Cell Biol. 78, 58-75.
GODEAU, F., SCHORDERET-SLATKINE, S., HUBERT, P. & BAULIEU, E. E. (1978). Induction of
maturation in xenopus laevis oocytes by a steroid linked to a polymer. Proc. natn. Acad.
Sci., U.S.A. 75, 2353-2357.
GOLBUS, M. S. & STEIN, M. P. (1976). Qualitative patterns of protein synthesis in the mouse
oocyte. /. exp. Zoo/. 198, 337-342.
JALABERT, B. (1976). In vitro maturation and ovulation in rainbow trout {Salmo gairdneri)y
northern pike (Esox lucius) and goldfish (Carassius auratus). J. Fish Res. Bd Can. 33,
974-988.
KAHNT, F. W. & NEHER, R. (1966). Uber die adrenale Steroid-Biosynthese in vitro. III.
Selektive Hemmung der Neben-nierenrinden-Funktion. Helv. chim. Acta 49, 725-732.
LlEBERMAN, M. E., TSAFRIRI, A., BAUMINGER, S., COLLINS, W. P., AHREN, K. & LlNDNER,
H. R. (1976). Oocytic meiosis in cultured rat follicles during inhibition of steroidogeneris. Acta endocr. (Kbh) 83, 151-157.
MASUI, Y. (1967). Relative roles of the pituitary, follicle cells and progesterone in the
induction of oocyte maturation in Rana pipiens. /. exp. Zool. 166, 365-375.
MCGAUGHEY, R. W. (1977). The culture of pig oocytes in minimal medium and the influence
of progesterone and oestradiol-17/? on meiotic maturation. Endocrinology 100, 39-45.
MOOR, R. M. (1977). Sites of steroid production in ovine Graaflan follicles in culture.
/. Endocr. 73, 143-150.
MOOR, R. M. (1978). Role of steroids in the maturation of ovine oocytes. Annls Biol. anim.
Biochim. Biophys. 18, 477-482.
MOOR, R. M., HAY, M. F., MCINTOSH, J. E. A. & CALDWELL, B. V. (1973). Effect of gonadotrophins on the production of steroids by sheep ovarian follicles cultured in vitro.
J. Endocr. 58, 599-611.
MOOR, R. M. & SMITH, M. W. (1978). Amino acid uptake into sheep oocytes. /. Physioi,
Lond. 284, 68-69P.
MOOR, R. M. & SMITH, M. W. (1979). Amino acid transport in mammalian oocytes. Expl
Cell Res. 119, 333-341.
MOOR, R. M., SMITH, M. W. & DAWSON, R. M. C. (1979). Measurement of intercellular
coupling between oocytes and cumulus cells using intracellular markers. Expl Cell Res.
(in the Press).
MOOR, R. M. & TROUNSON, A. O. (1977). Hormonal and follicular factors affecting maturation of sheep oocytes in vitro and their subsequent developmental capacity. /. Reprod
Fert. 49, 101-109.
MOOR, R. M. & WARNES, G. M. (1978). Regulation of oocyte maturation in mammals. In
Control of Ovulation (ed. D. B. Crighton, G. R. Foxcroft, N. B. Haynes & G. E. Lamming),
pp. 159-176. London: Butterworths.
O'FARRELL, P. H. (1975). High resolution, two dimensional electrophoresis of proteins.
J. biol. Chem. 250, 4007-4021.
SCHULTZ, R. M. & WASSARMAN, P. M. (1977). Specific changes in the pattern of protein
synthesis during meiotic maturation of mammalian oocytes in vitro. Proc. natn. Acad.
Sci., U.S.A. 74, 538-541.
SEAMARK, R. F., MOOR, R. M. & MCINTOSH, J. E. A. (1974). Steroid hormone production
by sheep ovarian follicles cultured in vitro. J. Reprod. Fert. 41, 143-158.
Steroid-oocyte interactions
335
L. D., ECKER, R. E. & SUBTELNY, S. (1968). In vitro induction of physiological
maturation in Rana pipiens oocytes removed from their ovarian follicles. Devi Biol.
17, 627-643.
SOUPART, P. (1974). The need for capacitation of human sperm; functional and ultrastructural observations. In Biology of Spermatozoa (ed. E. S. E. Hafez & C. Thibault),
pp. 182-191. Basle: Karger.
THIBAULT, C. (1977). Are follicular maturation and oocyte maturation independent processes?
/. Reprod. Fert. 51, 1-15.
THIBAULT, C , GERARD, M. & MENEZO, Y. (1975). Acquisition par l'ovocyte de lapine et
de veau du facteur de decondensation du noyau du spermatozoide fecondant (MPGF).
Annls Biol. anim. Bioch. Biophys. 15, 705-714.
TsAFRiRf, A., LIEBERMAN, M. E., AHREN, K. & LINDNER, H. R. (1976). Dissociation between
LH-induced aerobic glycolysis and oocyte maturation in cultured Graafian follicles of
the rat. Acta Endocr. 81, 362-366.
VAN BLERKOM, J. & MCGAUGHEY, R. W. (1978). Molecular differentiation of the rabbit
ovum. 1. During oocyte maturation in vivo and in vitro. Devi Biol. 63, 139-150.
WARNES, G. M., MOOR, R. M. & JOHNSON, M. H. (1977). Changes in protein synthesis
during maturation of sheep oocytes in vivo and in vitro. J. Reprod. Fert. 49, 331-335.
SMITH,
(Received 7 September 1979, revised 26 October 1979)