0021-972x/95/503.00/0 Vol. 80, No.

4
Journal of Clinical Endocrinology and Metabolism Printed in U.S.A.
Copyright 0 1995 by The Endocrine Society

Nongenomic Effects of 17P-Estradiol on Maturing

Human Oocytes: Relationship to Oocyte Developmental
Potential*
JAN TESARIK AND CARMEN MENDOZA
Center for Reproductive Biology and Medicine, American Hospital of Paris (J.T.), 92202 Neuilly sur
Seine; and Institut National de la Sante’ et de la Recherche Mbdicale (J.T.), 92140 Clamart, France;
and the Department of Biochemistry and Molecular Biology, University of Granada Faculty of
Sciences, Campus Universitario %uentenueua” (C.M.), 18071 Granada, Spain

ABSTRACT although the presence of extracellular Ca2+ still was an absolute

There is increasing evidence for nongenomic steroid effects in var- requirement for these [Ca”]i oscillations.
ious cell types. This study is the first to demonstrate that 17gestra- The addition of E, to oocyte maturation medium did not produce
diol (E,) exerts a direct nongenomic effect on maturing human oocytes any apparent effects on either germinal vesicle breakdown or further
by inducing a series of transient increases in the intracellular free progression of meiosis, but it did increase the fertilization and cleav-
calcium concentration ([Ca2’li). This effect of E, appeared to be spe- age rates of the in vitro matured oocytes.
cific and was mediated by steroid action on the cell surface. These findings show that E, can directly influence the quality of
The first of the E,-induced [Ca2’]i increases was induced by an maturing oocytes. This effect is due to steroid action on the cell
influx of extracellular Ca 2+ ions. However, the release of Ca2+ from surface, implies Ca2+ as a second messenger, and contributes to
intracellular stores contributed more significantly to [Ca’+Ji waves oocyte capacitation for fertilization and early postfertilization devel-
characterizing the subsequent series of secondary [Ca’+]i increases, opment. (J Clin Endocrinol Metab 80: 1438-1443, 1995)

I N NONMAMMALIAN species,oocyte maturation is un-

der direct hormonal control, and the effect of progester-
one (P4) on amphibian oocyte maturation was among the
generate or modulate this signal, similar to those in amphib-
ian oocytes, and so directly influence oocyte quality.
To test this hypothesis, we loaded human oocytes at the
earliest discovered nongenomic effects of steroids on cells(1). germinal vesicle (GV) stagewith a fluorescent Ca2+indicator
An immediate increase in the intracellular free Cazf con- and examined whether and how [Ca’+]i is influenced by the
centration [Ca”li isthe first detectable reaction of amphibian addition of E, to incubation medium. After preliminary ex-
oocytes to P, addition (2, 3). Mammalian oocytes do not periments demonstrating the development of [Ca2+]i oscil-
require an immediate hormonal stimulation to resume mei- lations in the oocytes in responseto E,, we replaced free E,
otic maturation (4). Notwithstanding, experience with hu- with a membrane-impermeant conjugate of this hormone to
man in vitro fertilization shows relationships between suc- decide whether hormone uptake into the cells was required
cessful conception and the concentrations of steroid to reduce this effect. We also treated oocytes with E, in
hormones in the fluid aspirated from follicles from which the CaE -free medium to determine the respective contributions
oocytes have been harvested (5-10). Although normal de- of intracellular and extracellular Ca2+ to the E,-induced
velopment of in vitro fertilized oocytes appears to be asso- [Ca2’li increases. Finally, experiments were performed to
ciated with a relatively high 17P-estradiol (E,)/androgen evaluate the effects of exposure of maturing oocytes to E, on
ratio in follicular fluid (lo), the mechanism underlying this subsequentmaturational events, fertilization, and early post-
relationship is not fully understood. fertilization development.
Moreover, spontaneous oscillations in [Ca2’li have re-
cently been shown to occur in mouse oocytes at the time of
germinal vesicle breakdown (GVBD), and it was suggested
that this Cazc signal may be important for later develo - Materials and Methods
mental processesrather than for GVBD itself (11). If a CaL Chemicals
signal generated during oocyte maturation creates a durable
imprint determining the developmental potential of the oo- E,, androstenedione (A), P,, BSA, E, 6-(O-carboxymethyl)oxime con-
jugated with BSA (30 mol E,/l mol BSA; E,-BSA), dimethylsulfoxide
cyte, it is possible that steroids present in follicular fluid (cell culture grade), light mineral oil (embryo tested), Dulbecco’s phos-
phate-buffered saline (PBS), 0.684 mmol/L ethylenediaminetetraacetic
Received October 5, 1994. Revision received December 16, 1994. Ac- acid (EDTA) in Ca’+- and Mg’+-free PBS (cell culture grade), and pro-
cepted December 28, 1994. tease from Streptomyces griseus (pronase) were purchased from Sigma
Address all correspondence and requests for reprints to: Dr. Jan (St. Quentin Fallavier, France). Sperm preparation medium (SPM) and
Tesarik, Center for Reproductive Biology and Medicine, American Has- hyaluronidase solution in SI’M were ol%a&ed from Medi-Cult (Copen-
pita1 of Paris, 63 boulevard Victor Hugo, 92202 Neuilly sur Seine, France. hagen, Denmark). B2 culture medium was obtained from BioMerieux
* This work was supported by Spanish Ministry of Education and (M&cy l’Etoile, France), and acetoxymethylester of flu03 (fluo3/AM)
Science Grant PB91-0713. from Molecular Probes (Eugene, OR).

1438

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 NONGENOMIC EFFECTS OF E, ON OOCYTES 1439

Gamete source and preparation surement. To minimize oocyte displacement by medium fluxes resulting
from stimulus addition, one or several oocytes were located on the
The oocytes used in this study were immature human oocytes aspi- microdrop periphery by manipulating them with the use of a fine glass
rated from antral ovarian follicles and still uossessinz an intact GV. All filament, where the oocytes were slightly compressed between the dish
of these oocytes came from cases in which oocytes w&e aspirated to be bottom and the mineral oil layer. The stimuli were then added with the
used for micromanipulation-assisted fertilization, and the identification use of a micropipette at the opposite side of the microdrop. With some
of oocytes with GV was enabled by removal of cumulus oophorus and practice, it was possible to add stimuli without disturbing the original
corona radiata cells, which is part of this procedure. Because of their position of oocytes, thus allowing a continuous measurement of [Ca2’]i.
immaturity, GV oocytes could not be used in the clinical protocol of In some cases, changes of medium were made outside of the confocal
micromanipulation-assisted fertilization; their use in the present exper- microscopy system, whereas the collection of images was interrupted for
imental protocol had institutional ethics board approval. a short time. Temporal changes in the fluorescence intensity recorded
Follicular aspiration was carried out under ultrasound control in from the oocytes were analyzed by using saved images and a Bio-Rad
cycles stimulated with hMG and hCG after ovarian desensitization with Time Course ratiometric Software Module.
a GnRH agonist (12). Oocyte-cumulus complexes were identified in
follicular aspirates and treated for l-2 min with the commercial solution
of hyaluronidase in SPM to remove the cumulus oophorus, followed by Oocyte in vitro maturation and fertilization
repeated aspirations into a finely drawn (internal diameter, 120 pm)
Pasteur pipette to remove the remaining corona radiata cells. Denuded All incubations were carried out at 37 C in B2 medium equilibrated
oocytes were then examined by phase contrast microscopy, and mei- with 5% CO, in air. Cumulus- and corona-free oocytes were incubated
otically mature (metaphase II) oocytes were subjected to subzonal in- for 24 h and inspected at regular time intervals for signs of oocyte
semination or intracytoplasmic sperm injection, using previously de- maturation (GVBD or extrusion of the first polar body). The medium was
scribed protocols (13,14). Those oocytes that still showed an intact GV supplemented with 2.6 X 10m3 g/L E,-BSA or the same concentration of
and lacked apparent signs of degeneration (such as granular aspect of BSA alone. This concentration of E,-BSA was chosen to corresuond I to
the cytoplasm or the presence of vacuole-like structures) were allocated 1 Fmol/L E,.
to this study with informed consent from the patients. A total of 210 GV Some oocytes that had achieved metaphase II by 24 h of incubation
oocytes were obtained, which represented 4.7% (210 of 4448) of all were inseminated in vitro with 1 X 105/mL spermatozoa. The sperma-
oocytes recovered in this group of patients. Some oocytes were further tozoa were prepared for in vitro insemination according to a standard
treated with a solution of pronase in PBS, as previously described (15), in vitro fertilization protocol (17). The inseminated oocytes were incu-
to remove the zona pellucida. These zona-free oocytes were preferen- bated with spermatozoa for 16 h. They were then washed in fresh
tially used in experiments in which the effect on E, on [Ca’+Ji was medium and examined for the presence of pronuclei. Oocytes showing
evaluated, whereas zona-intact oocytes were used in experiments in- two pronuclei were considered fertilized and were cultured for an ad-
volving oocyte in vitro maturation and fertilization. ditional 24 h to assess their capacity to undergo cleavage.
The spermatozoa used in this study were obtained from ejaculates of
healthy men with normal values of sperm density, motility, and mor-
phology (16). Spermatozoa were washed from seminal plasma and in- Statistical analysis
cubated for in vitro capacitation, as previously described (17). The time
of incubation in capacitating medium was 3-4 h. Percentages of oocytes that achieved individual developmental
stages in different treatment groups were compared by ,$ test.

Preparation and application of stimuli

E,, I’,, and A were dissolved in dimethylsulfoxide to a concentration Results
of 2 mmol/L. This solution was used to prepare solutions of the steroids
in SPM, which were used for direct additions to microdrops of SPM E2 acts at the oocyte plasma membrane to generate periodic
containing oocytes (see below). The concentrations of the steroids in transmembrane fluxes of extracellular Ca2+
these solutions were 4 times higher than those desired in the final
solution, and the volumes to be added were calculated according to the When [Ca”]i was recorded in GV stage oocytes without
actual volume of medium in the respective microdrop before each ad- any addition, no spontaneous fluctuations were observed.
dition. E,-BSA was dissolved in PBS to a concentration of 1 g/L, cor- Similarly, the addition of dimethylsulfoxide (solvent for un-
responding to 385 pmol/L E,. A second, 4-fold concentrated solution of
E,-BSA was then prepared in SPM and used as described for unconju-
conjugated steroids) up to a concentration of 128 mmol/L
gated E,. did not affect [Ca”]i. However, the addition of 1 pmol/L E,
to oocytes induced a transient increase in [Ca*‘li, followed
by a series of sharp secondary [Ca’+li increases ([Ca2’li
Evaluation of stimulus-induced changes in [Ca’+]i
oscillations; Fig. 1A). Each of the [Ca”li increaseslasted for
Relative changes in [Ca2’Ji were visualized with the use of a Bio-Rad lo-20 s, except for the first, which was of longer duration and
MRC 600 confocal laser scanning microscopy unit (Bio-Rad Laborato- usually took about 1 min (Fig. 1A). The interval between two
ries, Richmond, CA), by recording fluorescence emitted by oocytes sequential increaseswas rather constant for each oocyte and
loaded with fluo-3. Oocytes were loaded with this Ca2+ indicator by
incubating them for 30 min in SPM containing 25 pmol/L fluo-3/AM.
ranged between 20 s and 2 min in different oocytes. The
The loaded oocytes were transferred to microdrops (5 PL) of SPM duration of the whole series of [Ca2’]i oscillations lasted
contained in Falcon 3001 tissue culture dishes (Falcon, Oxnard, CA) and between l-6 h in individual cases.
covered with mineral oil. The dishes were placed on the heated stage of Both zona-intact and zona-free oocytes responded to E, in
a Nikon DiaFhot inverted microscope (Nikon Corp., Melville, NY),
where all [Ca +I1 measurements were made. Oocytes were scanned with
the same way. However, the lag period between hormone
a confocal argon ion laser beam with the use of a Nikon BHS filter addition and the first [Ca*‘]i increase was longer (30 s to 1
combination, and the resulting fluorescence images were saved in a min) in the caseof zona-intact oocytes compared with zona-
Panasonic l-gigabyte magnetooptical disk (Panasonic, Osaka, Japan) for free oocytes (lo-30 sl. That is why zona-free oocytes were
later analysis. The confocal plane was chosen to correspond to the used in most of the experiments involving [Ca2’]i measure-
equatorial plane of the oocytes. The scanning frequency was one image
each 2 sin most cases, but other frequencies, ranging between one image ment.
each second and one image each 5 s, were also used. Stimuli were added The observed effect of E, on [Ca2’li in oocytes appeared
to medium microdrops containing the oocytes in the course of mea- to be specific, becauseit could not be mimicked with even 10

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1440 TESARIK AND MENDOZA JCE & M . 1995
I’0180 . No 4

isting E,-induced lCa”li oscillations were promptly stopped

when oocytes were transferred to Ca’+-free medium and did
not reappear until they were put back into Ca*‘-containing
medium again (Fig. 2).
The site of E, action was located at the oocyte surface,
because E,-BSA, a plasma membrane-impermeant E, conju-
gate, was equally effective in producing the typical Ca2+
response as the free hormone (Fig. 3), whereas the addition
ii 0
0 2 4 6 of BSA alone, the macromolecular component of the conju-
gate, was without effect.
z 100
c
E2 sensitizes Ca’+-induced Cazf release from oocyte
5
E intracellular stores
.-
8 50 The spatial analysis of the temporal evolution of [Ca”]i
:
changes induced in oocytes by E,, as facilitated by confocal
::
2 microscopy, revealed a marked difference between the first
2 E,-induced [Ca”]i increase and the subsequent secondary
ii 0
0 2 4 6 8 10 12
[Ca*‘]i rises. The first [Ca’+]i increase was relatively slow
and occurred nearly simultaneously in the whole oocyte
Time (min)
cytoplasm, without signs of focal initiation or wave-like
FIG. 1. Effects of E, on [Caa+li in maturing human oocytes. Relative propagation (Fig. 4, A-D). In contrast, the second [Ca”li rise
changes in [Ca”li are expressed as changes in fluo-3 fluorescence was rapid and typically began in the oocyte periphery, from
intensity. A, Increase in [Caz+li and a series of secondary [Ca’+]i
oscillations after the addition of 1 pmol/L E, to a zona-free oocyte. B, which it spread as a Ca2+ wave throughout the rest of cy-
Absence of significant [Caa+li changes after sequential additions of 1 toplasm (Fig. 4, E-H). All subsequent [Ca2’]i increasesfol-
and 10 pmol/L A to a zona-free oocyte, followed by the development lowed the same spatial propagation pattern. Of course, the
of [Ca2+1i oscillations after subsequent addition of 1 Fmol/L E,. The propagation of the Ca2+waves could only be traced in those
Caa+ responses shown in A and B are representative of those observed
in other 11 and 9 oocytes, respectively.
series of confocal images in which the plane of vectorial
propagation of the wave front coincided, at least roughly,
with the confocal plane at which the images were taken.
times higher concentrations of A (Fig. 1B) and I’, (data not
BecauseCa’+-induced Ca2+release(CICR) is the mechanism
shown).
underlying the propagation of Ca2+waves in cells (18), these
Both the initial [Ca”]i increase induced by E, and the
observations mean that when undergoing the first E,-in-
subsequent series of [Ca*‘]i oscillations were strictly depen- duced [Ca2+]i rise, oocytes still have a limited capacity to
dent on the presence of Ca2+ in the extracellular space. In
activate CICR, but this capacity increasesmarkedly by the
Cazf-free medium (0.684 mmol/L EDTA in Ca’+- and Mgzc- time of the second rise.
free PBS), the addition of E, did not produce anr effect on
[Ca”li, but subsequent transfer of oocytes to Ca +-contain-
Exposure of maturing oocytes to E2 does not affect
ing medium in the continuous presence of E, caused an
progression of meiosis, but improves developmental
immediate typical Ca2+ response (Fig. 2). Furthermore, ex-
potential of oocytes

Ca-free Ca Ca-free Ca When GV oocytes were incubated in vitro in B2 medium

supplemented either with 2.6 X 1O-3 g/L E,-BSA, corre-
sponding to 1 Fmol/L E,, or with 2.6 X 1O-3 g/L unconju-
gated BSA, equivalent proportions (P < 0.05) of oocytes
resumed meiosis by 6 h of incubation and achieved meta-

cZ- 100 , E2-BSA

0 4 8 12 16 20 24

Time (min)

FIG. 2. Dependence of [Ca2’]i oscillations induced by the addition of =

1 pmol/L E, to a zona-free oocyte on the presence of Caa+ ions in the IL 0
extracellular space. In the continuous presence of the steroid, the 0 2 4 6
Ca2+ response is reversibly inhibited in Ca2+- and M$+-free PBS
Time (min)
containing 0.02% EDTA (Ca-free) and immediately restored in me-
dium containing 1.7 mmol/L Ca2+ (Cal. Short nans in the record occur FIG. 3. Caa+ response induced by the addition to a zona-free oocyte
at the times ofmedium changes, when data collection was briefly of Ez-BSA (1 pmol/L E,), a membrane-impermeant E, conjugate. The
interrupted. The Ca2+ response shown is representative of those Ca2’ response shown is representative of those observed in other
observed in seven other oocytes. seven oocytes.

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 NONGENOMIC EFFECTS OF E, ON OOCYTES

FIG. 4. Spatia uman oocyte. The i cha ssed in pseudocolor

according to the scale bar, where the lowest values are coded black. A-D, Rising phase of the initial [Ca2+]i increase after E, addition to a
zona-free oocyte. The time interval between successive images displayed in this series is 4 s (every fourth image from a series of recordings
taken at l-s intervals is shown). E-H, Rising phase of the first of the series of secondary [C!az+]i elevations after the initial Ez-induced [Ca2’]i
increase. The time interval between successive images displayed in this series is 1 s.

phase II by 24 h of incubation in both groups (Fig. 5). How- q E2+BSA

ever, the fertilization and cleavage rates of metaphase II 0 BSA
oocytes that matured in the presence of E,-BSA were higher
than those of oocytes that had matured in medium supple-
mented with BSA alone (Fig. 6). These data show signifi-
cantly higher percentages of oocytes developing two pro-
nuclei (P < 0.05) and of those undergoing at least one
cleavage division (P < 0.01) in the E,-BSA group.

Discussion

Nearly all of the major steroid hormone classeshave been

demonstrated to have nongenomic effects (19). After the
early reports on rapid P4 effects on frog oocytes (’l-3), sim- MI 2PN Cleavage
Developmental stage
76) E2-BSA FIG. 6. In vitro fertilization and cleavage results with human oocytes
BSA matured ilz vitro in the presence of membrane-impermeant E, con-
jugate (E,-BSA) at a concentration corresponding to 1 pmol/L E,
compared with those using oocytes matured in control medium (sup-
-59 plemented with BSA only). The percentages of two-pronucleated oo-
cytes (2PN) and those that underwent at least one cleavage division
(Cleavage) were determined 16 and 48 h after insemination, respec-
tively. The numbers ofoocytes are giveninparentheses above the bars.

ilarly rapid effects have been described for I’, in spermatozoa

(20-22) and neurons (23,24), for lcY,25-dihydroxyvitamin D,
in osteoblasts(25,261and enterocytes (27,281,for aldosterone
in mononuclear leukocytes (29-31), for glucocorticoids in
vascular smooth muscle cells (32,331,and for ring A-reduced
- I’, and deoxycorticosterone steroids in neurons (34).
Most of the known nongenomic effects of steroids on cells
GV MI
are related to an increase in [Ca*‘]i. However, there is a
Maturation stage considerable variability in the mechanism of steroid-induced
FIG. 5. In vitro maturation of human GV oocytes incubated in the Ca2+ mobilization and the source of the mobilized Ca*+. In
presence of membrane-impermeant E, conjugate (E,-BSA) added at this study, the initial [Ca*‘]i increase occurring in maturing
a concentration corresponding to 1 PrnolL E, compared with that of human oocytes in response to E, is shown to be induced by
oocytes matured in control medium (supplemented with BSA only).
The percentages of metaphase I (MI) and II (MID oocytes were de-
an influx of extracellular Ca*+. The same effect can be pro-
termined after 6 and 24 h of incubation, respectively. The numbers of duced by a membrane-impermeant E, conjugate. In this re-
oocytes are given in parentheses above the bars. spect, the action of E, on human oocytes is analogous with

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1442 TESARIK AND MENDOZA JCE & M . 1995
Vol80. No 4

the action of I’, on human spermatozoa (35, 36), but quite cillations in maturing human oocytes is another question. The
different from the previously described nongenomic action absenceof noticeable effects of the presenceof E, in medium
of E, on chicken, pig, and rat ovarian granulosa cells, in usedfor oocyte in vitro maturation on the progressionof meiosis
which E, increases [Ca”]i by producing a rapid release of suggeststhat neither the presenceof E, nor [Ca2’]i oscillations
Ca2+ from intracellular stores (37). are necessaryfor oocyte meiotic maturation. This is in agree-
The ability of E, to induce a seriesof secondary [Ca”]i ment with the previous observation that abolishing the spon-
oscillationsafter the primary [Ca”li rise alsodistinguished the taneous [Ca2’li oscillations of immature mouse oocytes does
responseof human oocytes to the hormone (this study) from not affect GVBD (11). On the other hand, the improved fertil-
that of granulosa cells,in which only a single transient [Ca”]i ization and cleavage rates of the oocytes exposed to E, during
surge occurred (37). The propensity to generate [Ca”]i oscil- the early stagesof maturation compared to control values sug-
lations in responseto different stimuli is a known property of gestthat the action of E, supports the cytoplasmic maturational
mammalian oocytes. In fact, [Ca”]i oscillations have been changesnecessaryfor fertilization and early postfertilization
shown to representan early reaction of oocytesto the fertilizing development. A relative independence of the developmental
spermatozoonin various mammalian species(reviewed in Ref. processescontrolling nuclear and cytoplasmic maturation of
38), including the human (14, 39, 40). Oscillations of [Ca’+]i mammalian oocytes has been sufficiently documented (re-
have alsobeen found to develop spontaneouslyduring in vitro viewed in Ref. 45).
maturation of murine oocytes (11).In contrast, no spontaneous Interestingly, previous work hasshown that human oocytes
[Ca2’]i oscillationswere observedto occur in human oocytes in isolated from follicles with undetectable levels of E, can un-
this study. dergo fertilization and embryonic cleavage in vitro (46). How-
Interestingly, the spatial analysis of E,-induced [Ca2’]i ever, those observations were made in a woman with congen-
risesshowed that when undergoing the first [Ca2’]i increase ital adrenal hyperplasia due to 17a-hydroxylase deficiency in
after E, addition, the oocyte still has a limited ability to whom, consequently, all 17-hydroxylated steroids, including
activate CICR in responseto E,-induced Ca2+entry, whereas androgens,were affected. In view of the fact that oocyte quality
the typical attributes of active CICR, a wave-like propagation seemsto be better correlated with the E,/androgen ratio than
and amplification of the zone of increased [Ca2+]i, were with the absolute level of each of them (lo), it is possiblethat
present beginning with the second lCa*‘]i rise. The activa-
the E, action is needed to protect oocytes from the atresia-
tion of CICR entails a more significant contribution of in-
promoting effect of follicular androgens.
tracellularly stored Ca2+to the secondary E,-induced [Ca2’]i
It also hasto be noted that the oocytesusedin this study were
rises, although the presence of extracellular Ca2+ was an
still at the GV stage and, thus, remained behind the oocytes
absolute requirement for the maintenance of [Ca2’]i oscil-
obtained at the sametime from the most advanced follicles that
lations in our system. In this regard, the E,-induced [Ca2’]i
already were at metaphaseII. Such oocytes, also termed non-
oscillations described in this study were similar to the spon-
ovulatory, have been shown previously to lack ultrastructural
taneous [Ca*‘]i oscillations observed in maturing mouse
oocytes (11). degenerative changes,to synthesize proteins, and to resume
meiosisin vitro under appropriate culture conditions (47). They
Why the CICR response becomes more active in human
oocytes after the first E,-induced [Ca*‘]i increase is an in- must thus be distinguished from atretic oocytes in which irre-
triguing question that warrants further investigation. One versible degenerative changeshave already started. Neverthe-
possible explanation is that the first [Ca2+]i rise somehow less,further work is necessaryto determine whether GV oo-
awakens a dormant intracellular Ca*+-releasesystem, sothat cytes obtained from small antral follicles at the time of the
the contribution of intracellularly stored Ca2+ ions augments ovarian cycle at which more advanced healthy follicles are not
the following [Ca”]i increases.On the other hand, the ac- yet present in the ovary behave in the sameway.
tivation of CICR may also be due to superimposition of a Even though this pilot study leaves many questions con-
Ca2+-independent action of E, rather than to the primary cerning the mechanism of E, action on human oocytes still
E,-induced [Ca2’]i surge. Such a dual nongenomic action is open, this is the first time that it hasbeen suggestedthat oocyte
supposed to mediate the effect of I’, on human spermatozoa, quality may be conditioned by a direct nongenomic action of
in which I’, appears to simultaneously open a plasma mem- follicular steroid. It has been shown that the developmental
brane Ca2+ channel (36), possibly by acting primarily on a potential of human oocytes is related to the E,/androgen ratio
y-aminobutyric acid,-like receptor/Cl- channel (41), and to in follicular fluid rather than to the absolute E, concentration
activate, in a Ca2’-independent manner, a protein tyrosine (10). It would thus be of interest to examine whether and how
kinase (22). Becausethe best-establishedmechanism of CICR the nongenomic response of oocytes to E, is influenced by
in mammalian oocytes involves inositol trisphosphate (IF’,)- different concentrationsof androgensand whether the presence
sensitive Ca2+ stores (42, 43), and IF’, can be generated by of androgen during oocyte in vitro maturation counteractsthe
phospholipase-Cy activatable by a tyrosine kinase (44), si- beneficial effect of E, on oocyte developmental potential. These
multaneous activation by E, of a plasma membrane Ca2+ experiments are under way in our laboratories.
channel and a tyrosine kinase might stimulate Ca*+ influx
and CICR, respectively. It remains to be elucidated whether
E, actually stimulates II’, production in human oocytes and,
if so, whether this effect is mediated by the E,-induced Ca2+ Acknowledgment
influx or by an independent E, action. The technical assistance of Ms. Beatrice Berdrin is gratefully acknowl-
The physiological significanceof the E,-induced [Ca*‘]i os- edged.

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 NONGENOMIC EFFECTS OF E, ON OOCYTES 1443

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