17{beta}-Estradiol and progesterone improve in-vitro cytoplasmic maturation of oocytes from unstimulated prepubertal and adult rhesus monkeys

Ping Zheng1,2, Wei Si1,2, Barry D. Bavister2,3, Jifeng Yang1,2, Chenghui Ding1,2 and Weizhi Ji1,2,4

1 Department of Primate Biology, 2 China–US Primate Biology Laboratory, Kunming Institute of Zoology, Chinese Academy of Sciences, 32 Jiao Chang Dong Lu, Kunming 650223, China and 3 Department of Biological Sciences, University of New Orleans, and Audubon Center for Research of Endangered Species, New Orleans, Louisiana, USA

4 To whom correspondence should be addressed. e-mail: wji{at}mail.kiz.ac.cn


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Effects of 17{beta}-estradiol and progesterone on rhesus monkey oocyte maturation in vitro were evaluated by embryo development subsequent to IVF. METHODS AND RESULTS: In experiment 1, immature cumulus–oocyte complexes collected from unstimulated adult females during the non-breeding season were matured in modified medium CMRL-1066 containing various combinations of gonadotrophins (FSH + LH), estradiol and/or progesterone. Formation of morulae and blastocysts was greatest in oocytes matured in medium containing estradiol and/or progesterone, with or without gonadotrophins (morula 38–46%, blastocyst 14–20%) than in control oocytes matured without estradiol or progesterone (morula 14%, blastocyst 0%). In experiment 2, cumulus–oocyte complexes from unstimulated prepubertal female monkeys were matured in medium with gonadotrophins, estradiol or progesterone. The best development to the morula stage was obtained with oocytes matured with gonadotrophins and estradiol or gonadotrophins and progesterone (43 and 25 morulae, respectively), while control oocytes matured with gonadotrophins but without steroid hormones gave the poorest morula developmental response (12%). However, there was no difference in blastocyst development across all groups (0–3%). CONCLUSIONS: These results demonstrate that during rhesus monkey oocyte maturation in vitro: (i) estradiol or progesterone can improve oocyte developmental competence; (ii) immature oocytes from prepubertal versus adult females have differential responses to challenge with estradiol or progesterone.

Key words: embryo development/17{beta}-estradiol/oocyte maturation in vitro/progesterone/rhesus monkey


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Primate (human and monkey) IVF clinical and research programmes are generally performed using oocytes from donors stimulated with exogenous gonadotrophins. In humans, this treatment carries the risk of ovarian hyperstimulation syndrome with serious adverse effects, and it is contraindicated in patients with polycystic ovarian syndrome (Trounson et al., 1994Go; Cha and Chian, 1998Go; Cha et al., 2000Go). In addition, the gonadotrophin preparations used for patient stimulation are very expensive. To reduce or circumvent these problems, in-vitro maturation (IVM) of oocytes together with IVF has been employed in both low-dose FSH-stimulated and in natural (unstimulated) cycles (Paulson et al., 1994Go; Barnes et al., 1996Go; Cha and Chian, 1998Go; Suikkari et al., 2000Go). However, primate oocytes, when collected from unstimulated or even from FSH-stimulated females, that are matured and fertilized in vitro have reduced or very poor developmental competence compared with oocytes matured in vivo (Morgan et al., 1991Go; Schramm and Bavister, 1996Go, 1999Go; Gilchrist et al., 1997Go; Cha et al., 2000Go; Zheng et al., 2001aGo) or IVM oocytes of other mammals such as mice, cattle and sheep (Schroeder and Eppig, 1984Go; Sirard et al., 1988Go; Galli and Moor, 1991Go). This might be due to poor ‘cytoplasmic maturation’ resulting from suboptimal culture conditions (Eppig et al., 1994Go; Eppig, 1996Go; Krisher and Bavister, 1998Go; Schramm and Bavister, 1999Go). Therefore, better understanding of factors such as hormones, growth factors, energy substrates and nutrients that are important for oocyte maturation will improve embryonic development of IVM oocytes in primates and other species (Rose-Hellekant et al., 1998Go; Schramm and Bavister, 1999Go; Abeydeera et al., 2000Go; Spindler et al., 2000Go). It is noteworthy that, to date, if only oocytes from donors that were not stimulated with hCG are considered as undergoing maturation completely in vitro, one non-human primate offspring and only a small number of human babies have been born following transfer of embryos derived from IVM (e.g. Cha et al., 2000Go; Schramm and Paprocki, 2000Go).

In vivo, oocyte growth and maturation are directly regulated by intraovarian factors such as steroids, cytokines and other growth factors acting at key points during the process of follicle development (Campbell and McNeilly, 1996Go; Fulvio, 1996Go). Among these factors, estradiol (E2) may be of great importance. In non-primate species such as the rabbit, bovine and hamster, E2 is required for follicular development and maturation in vivo (Richards, 1980Go) and also for oocyte fertilization (Suzuki et al., 1984Go; Yoshimura et al., 1986Go; Saeki et al., 1991Go). In contrast to several non-primate species, primates do not require high or increasing E2 levels for follicle growth and oocyte meiotic (nuclear) maturation (Zelinski-Wooten et al., 1993Go, 1994Go). However, E2 may be important in regulating oocyte cytoplasmic maturation as it is required for acquisition of monkey oocyte fertilization competence (Zelinski-Wooten et al., 1994Go). Successful pregnancies were more likely to occur in human IVF using oocytes from follicles that contained higher concentrations of E2 and a higher E2:progesterone ratio (Carson et al., 1982Go). However, Morgan et al. (1990Go) did not find any correlation between rhesus monkey follicular steroid concentrations and IVF success, while lower E2 levels and higher progesterone:E2 ratios were associated with faster-cleaving embryos. Thus, in primates the absolute level of E2 may be most important for supporting oocyte cytoplasmic maturation. Estradiol was found to act directly at the human oocyte surface during IVM to improve fertilization and cleavage (Tesarik and Mendoza, 1995Go). Taken together, these data suggest that E2 may be involved in oocyte embryonic developmental competence, in ways that remain to be clarified. However, this role of E2 is not universal among animals because it inhibited oocyte maturation in frogs (Lin and Schuetz, 1983Go).

Progesterone is another intrafollicular steroid mediator of normal mammalian ovarian function. Progesterone is required for ovulation, fertilization, luteinization and maintenance of luteal structure and function in rats and monkeys (Armstrong et al., 1991Go; Hibbert et al., 1996Go). However, with respect to the participation of progesterone in oocyte maturation, little is known in primates or even in rodents, despite limited evidence that levels of progesterone in follicular fluid are closely associated with oocyte maturity and quality. For instance, in humans and rhesus monkeys, high ratios of progesterone to E2 in follicular fluid were correlated with superior embryonic development and pregnancy frequency (Basuray et al., 1988Go; Morgan et al., 1990Go). In amphibians and fish, it has been known for many years that progesterone or its analogues induces oocyte maturation, acting at the surface of the oocyte rather than by the conventional genomic route (Sadler and Maller, 1982Go; Baulieu and Schorderet-Slatkine, 1983Go; De Albuja et al., 1983Go; Nagahama and Adachi, 1985Go; Morrison et al., 2000Go).

In our laboratory, we have established chemically defined conditions for IVM of rhesus monkey oocytes that can undergo embryo development following IVF (Zheng et al., 2001aGo). This serum-free culture system allows examination of hormone actions without complications from undefined serum factors (Bavister, 1995Go). Therefore, the present study was designed to test possible roles of E2 and progesterone on the meiotic and developmental competence of rhesus monkey oocytes during IVM as judged by IVF followed by in-vitro culture of embryos.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals, treatments and oocyte recovery
During the non-breeding season (March–August in Kunming, China), 19 and 14 pairs of ovaries, respectively, were collected from healthy prepubertal (1–2 year old) and adult (5–15 year old) rhesus monkeys, which had never been used for any experiment. These normal animals were euthanized by another institute to provide kidney cells for polio vaccine production. The adult females which showed normal menstrual cycles in the breeding season exhibited no menstruation after March. The ovaries were collected into TALP-HEPES medium (Bavister et al., 1983aGo) and transferred to the laboratory at ~30°C within 1–2 h after removal. Antral follicles >=1000 µm in diameter were dissected from the excised ovaries and processed as described previously (Schramm et al., 1993Go). Follicles showing obvious atresia (dark, dispersed granulosa cells) were discarded. Remaining follicles were held for 2–6 h in the same medium at 37°C while the oocytes from antral follicles were being collected, then they were punctured for retrieval of germinal vesicle (GV) stage oocytes.

Oocyte in-vitro maturation
Oocytes were used that appeared normal (round and medium to lightly pigmented), contained an intact nuclear membrane (GV stage) and were enclosed by at least two layers of tightly condensed cumulus cells (cumulus–oocyte complexes, COC). The normal COC were randomly grouped and washed four times in their respective culture medium before IVM. In order to sustain the concentrations of steroids in the media drops, 4 ml of mineral oil were incubated with 4 ml of mCMRL-1066 containing no steroid (control) or supplemented with E2 (100 ng/ml, from 50 µg/ml of E2 stock constructed in absolute alcohol) and/or progesterone (3 µg/ml, from 1.5 mg/ml of P4 stock constructed in absolute alcohol) for 3 days. For each of the four IVM culture treatments (control, E2, progesterone, E2 + progesterone; see below), nine 50 µl drops of medium were put into a separate 35 mm plastic Petri dish and covered with 3.5 ml of the corresponding steroid-equilibrated oil and incubated at 37°C for ~3 h before addition of oocytes. Five to 10 COC were then placed in several culture drop(s) of each treatment and cultured for 32–36 h at 37°C under 5% CO2 in air with 100% humidity. It is likely that COC produce very small amounts of E2 and progesterone during culture with gonadotrophins. To control for any effects of such endogenous steroids, one treatment was included with gonadotrophins but no added steroids. In this study, the purpose was to examine effects of steroid hormones on oocyte developmental competence, and not to directly assess effects on COC function. In five replications of experiment 1 across days, a total of 325 normal-looking COC retrieved from 14 adult rhesus monkey ovaries were matured in: (1) control medium: modified CMRL-1066 (Gibco, NY, USA) (mCMRL-1066) + ovine FSH (5 µg/ml; oFSH-NIADDK-NIH, AFP55518) + ovine LH (10 µg/ml; oLH-NIADDK-NIH, AFP4117); (2) mCMRL-1066 + FSH + LH + E2 (100 ng/ml; Sigma Chemical Co., USA); (3) mCMRL-1066 + FSH + LH + progesterone (3 µg/ml; Sigma); (4) mCMRL-1066 + FSH + LH + E2 + progesterone; and (5) mCMRL-1066 + E2 + progesterone. Medium CMRL was modified by addition of lactate, pyruvate and glutamine (Boatman, 1987Go) and contained no macromolecular supplement. A total of 228 normal-looking immature COC were collected from 19 prepubertal female monkeys and used for five replicates across days of experiment 2 in which the COC were cultured in: (1) control medium: mCMRL-1066 + FSH + LH; (2) mCMRL-1066 + FSH + LH + E2; and (3) mCMRL-1066 + FSH + LH + progesterone. Because the availability of ovaries from adult versus prepubertal animals could not be controlled, experiments 1 and 2 were carried out in parallel rather than sequentially.

Oocyte IVF and embryo culture
In all treatments, oocytes that extruded a first polar body (metaphase II, MII) were subjected to the same IVF and embryo culture procedures. Frozen–thawed semen was washed then sperm capacitation and IVF were conducted as described previously (Bavister et al., 1983bGo). Capacitated sperm were co-incubated with the respective treatment groups of MII oocytes for 12–16 h at 37°C in a humidified atmosphere of 5% CO2 in air. Adhering sperm and cumulus cells were then removed manually by passing the oocytes through a pulled glass pipette. Oocytes were examined with Nomarski optics for evidence of activation (containing two polar bodies and/or >=1 pronucleus). All oocytes containing three or more pronuclei were considered polyspermic and excluded from the fertilization data.

Activated oocytes were washed and cultured in 50 µl drops of mCMRL-1066 (5–10 per drop) containing 20% bovine calf serum (BCS) at 37°C under oil in a humidified atmosphere of 5% CO2 in air until development was arrested or until ‘hatching’ (escape from the zona pellucida) occurred. The oocytes were transferred to fresh embryo culture medium every other day. Embryos were examined daily using Nomarski optics (x200–400 magnification) on a Nikon Diaphot TMD microscope. Embryos with a pronounced blastocoele cavity were classified as blastocysts.

Measurement of steroid hormone concentrations in culture media under oil
Sets of mCMRL-1066 culture drops under oil like those used for the IVM experiments were prepared containing the same concentrations of E2 or progesterone. An enzyme immunoassay (EIA) as described by Lorenzo et al. (1997Go) was used to measure concentrations of these steroids remaining in the culture media drops at intervals during incubation for up to 32 h. These measurements were made on three separate occasions. The kit for E2 measurement is ActiveTM Estradiol EIA, DSL-10-4300 (Diagnostic Systems Laboratories, Inc., USA) and the one for progesterone is ActiveTM Progesterone, DSL-10-5000 (Diagnostic Systems Laboratories).

Data analysis
Values for MII were analysed as the percentages of total oocytes, and values for activated oocytes and all stages of embryos were analysed as the percentages of MII oocytes. All percentage data were subjected to arcsin (square root) transformation. The transformed data were analysed by one-way analysis of variance and Fisher’s protected least significant difference (LSD) test. Values with P < 0.05 were considered statistically significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Concentrations of steroid hormones remaining in the culture media drops during culture are shown in Tables I and II. The concentration of E2 fell by ~50% during the first 4 h but levelled off from 8 h onwards to ~35 ng/ml (Table I). In contrast, progesterone concentrations fell dramatically over the first 2 h to only 7% of their original level, then decreased much more slowly until they were stable by 16 h at a little less than 2% of the starting concentration (Table II). Because oocytes were added ~3 h after the start of equilibration of the culture media drops with steroid-equilibrated oil, the concentration range of E2 in the culture medium during oocyte culture for 32–36 h was therefore ~76–35 ng/ml, and for progesterone the range was ~185–45 ng/ml over this time period.


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Table I. Concentrations of estradiol (E2) in culture medium (mCMRL-1066) overlaid by E2-equilibrated oil at different incubation time points
 

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Table II. Concentrations of progesterone in culture medium (mCMRL-1066) overlaid by progesterone-equilibrated oil at different incubation time points
 
Results of IVM experiment 1 are shown in Table III. Overall, oocytes across all treatments showed similar percentages of meiotic maturation, fertilization and embryo development up to the 8-cell stage (P > 0.05 non-significant). However, oocytes matured in groups 2–5 with different combinations of E2 and progesterone displayed superior development to the morula and blastocyst stages compared to those in the control group without any steroid hormones in which there were no blastocysts (P < 0.05). However, there were no treatment differences among groups 2–5. In treatment groups 2–5, 40, 67, 43 and 40%, respectively, of blastocysts expanded (data not shown in Table III). The time intervals for blastocyst formation were also similar across all treatment groups: 153–178 h after insemination. Because all blastocysts were used for other purposes, the hatching and the cell number of blastocysts could not be determined in this study.


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Table III. Effects of estradiol (E2) and progesterone with or without gonadotrophins on meiotic and developmental competence of oocytes from unstimulated adult rhesus monkeys during the non-breeding season
 
In experiment 2 (Table IV), no significant differences were found in oocyte meiotic maturation, fertilization and embryo development prior to the 8-cell stage among the three treatments (P > 0.05 non-significant). However, oocytes matured in treatment containing E2 (treatment 2) showed better development to >=8-cell and to morula stage than those in control medium (no steroid hormone, treatment 1). Oocytes in treatment containing progesterone (treatment 3) also displayed superior development into morulae than the control (P < 0.05). There was no significant difference in embryo development between treatments 2 and 3. Only one blastocyst formed in treatment 2.


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Table IV. Effects of estradiol (E2) and progesterone on meiotic and developmental competence of oocytes from unstimulated prepubertal rhesus monkeys
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Several studies on the local modulation of primate oocyte maturation by steroids have involved in-vivo depletion of E2 and/or progesterone (Zelinski-Wooten et al., 1993Go, 1994Go; Hibbert et al., 1996Go), in-vivo treatment with pharmacological doses of steroids (Richards, 1980Go; Koering et al., 1991Go) or investigation of correlations between follicular fluid steroid content versus oocyte maturity and quality (Basuray et al., 1988Go; Morgan et al., 1990Go). However, these studies are all indirect and interpretation of responses therefore might be distorted, e.g. by effects of steroid depletion or accumulation on ovarian function that are not directly related to oocyte biology. In the present study, we directly examined the role of steroids on oocyte competence by maturing COC in vitro, using a chemically defined culture medium. Additionally, COC from unstimulated prepubertal and adult rhesus monkeys obtained during the non-breeding season, which have low levels of E2 and progesterone (Walker et al., 1984Go), were used to avoid in-vivo actions of high levels of E2 and progesterone on oocytes prior to oocyte collection.

The problem of absorption of steroids by the oil overlaying the culture media drops was addressed by equilibrating the oil with the appropriate steroid and by measuring steroid hormone concentrations in the culture medium at intervals during incubation. The concentrations of E2 and of progesterone in the medium reached equilibrium at ~8 and ~16 h, respectively. Although the concentration of progesterone decreased much more rapidly than that of E2, the final concentrations were about the same, i.e. 35–48 ng/ml. These concentrations are similar to that for progesterone (32 ng/ml) but far lower than for E2 (400 ng/ml) in rhesus monkey follicular fluid at the end of 8 days of FSH stimulation (Chaffin et al., 1999Go). Nevertheless, the in-vivo concentrations of these hormones are likely to be much greater than those required for their actions on the cumulus/granulosa cells because of the need to maintain adequate systemic steroid hormone levels. Conversely, even though the level of progesterone in the culture drops decreased very rapidly, there was still enough of this steroid to exert a detectable effect on oocyte maturation, as discussed below. By using a high concentration of progesterone at the beginning of incubation (almost 3000 ng/ml), it appears that the capacity of the oil overlay to absorb this steroid was close to being saturated before the oocytes were added, thus sustaining an adequate concentration of between 211 and 48 ng/ml for the duration of oocyte culture. As discussed later, steroid hormones may act very rapidly, through mechanisms including non-genomic pathways, so that the higher concentrations of E2 and progesterone present towards the beginning of oocyte culture (starting 3 h after steroid incubation began, Tables I and II) were sufficient to exert their actions on oocyte maturation, as demonstrated by the data.

For the first time, our data provide evidence that E2 and progesterone significantly improve the developmental competence (cytoplasmic maturation) of primate oocytes during IVM. However, in both experiments 1 and 2, COC matured in all treatments (i.e. with or without steroid hormones present) completed meiotic maturation at similar frequencies, indicating that E2 or progesterone at the levels we used does not alter nuclear maturation of rhesus monkey oocytes. This result is consistent with reports that impairment of steroid biosynthesis in adult monkeys did not affect oocyte meiotic status (Zelinski-Wooten et al., 1994Go; Hibbert et al., 1996Go). Oocyte nuclear maturation was also unaffected after exposure of hamster oocytes to trilostane in vitro (Suzuki et al., 1984Go) and of rabbit ovaries to an inhibitor of cholesterol side-chain cleavage enzyme (Yoshimura et al., 1986Go). However, data inconsistent with our results were reported by Gould and Graham (1976Go) also using prepubertal rhesus monkey females. In that study, progesterone improved the frequency of attaining MII, but cytoplasmic maturation of oocytes was not examined. The discrepancy between these two studies with respect to progesterone could be related to several key experimental differences. In the study by Gould and Graham, oocytes were collected after females had been stimulated with equine chorionic gonadotrophin for 5 days, and cumulus/granulosa cells enclosing oocytes were removed prior to IVM. In addition, Ham’s F-10 medium + fetal calf serum was used as the IVM culture medium, while progesterone was used at 25 µg/ml versus 3 µg/ml in our study, and the medium concentration may have been much higher due to steroid binding by serum proteins. Yet another difference is that progesterone activity on nuclear maturation was examined in combination with dbcAMP by Gould and Graham (1976Go).

It should be also noted that in the present study and other related reports, in-vivo production of E2 and progesterone could not be completely ruled out (Koering et al., 1991Go; Zelinski-Wooten et al., 1993Go, 1994Go; Hibbert et al., 1996Go), so there could have been some in-vivo priming of COC with endogenous steroids prior to collection. Therefore, we cannot eliminate the possibility that oocyte nuclear maturation in rhesus monkeys requires a very low level of E2 or progesterone.

Several studies have defined the requirement of E2 or progesterone for the acquisition of oocyte fertilizability. For example, experimental reduction of E2 and progesterone production during the follicular phase impaired fertilization of rhesus monkey oocytes (Zelinski-Wooten et al., 1994Go). Estradiol and/or progesterone also play an important role in supporting normal fertilization in the rat (Zhang and Armstrong, 1989Go), hamster (Suzuki et al., 1984Go) and rabbit (Yoshimura et al., 1986Go). However, our data showed no effect of exogenous E2 or progesterone during IVM on the fertilizability of rhesus monkey oocytes across all treatments in experiments 1 and 2. Similarly, Morgan et al. (1990Go) and Basuray et al. (1988Go) found no relationship between the follicular fluid steroid (E2 and progesterone) content and oocyte fertilization in rhesus monkey and human. It is possible that low endogenous levels of E2 and progesterone in the monkeys used in this study are adequate to support cytoplasmic events in oocytes that are required for the occurrence of fertilization. If so, we would not expect to find an additional effect of steroids in the IVM culture medium on fertilization.

Consistent with the present study, few (4%) or no blastocysts were produced from IVM oocytes obtained during the non-breeding season from adult rhesus monkeys, whether the animals were FSH-stimulated or not; and IVM oocytes from unstimulated animals during the breeding season also did not develop into blastocysts (Zheng et al., 2001bGo). The poor development of some IVM oocytes past the 8-cell stage in some conditions may be related to improper activation of the embryonic genome due to defective maturation (Schramm et al., 2003Go). Only when monkeys were stimulated with FSH during the breeding season (Zheng et al., 2001aGo,b) did their IVM oocytes produce blastocysts at a level (16–30%) similar to that obtained in the present study. In both of these studies (Zheng et al., 2001aGo,b), no E2 or progesterone was used in the IVM culture medium. The blastocyst development results obtained in these studies and in the present work could be reconciled by a need during oocyte maturation for steroid hormones that are either produced in vivo under the influence of FSH (to which monkeys respond readily only in the breeding season) or supplied in vitro during oocyte culture, as in the present study. Inclusion of steroid hormones in the culture medium for IVM supports cytoplasmic maturation and allows use of oocytes from unstimulated rhesus monkeys for research throughout the year, which is advantageous in view of the prolonged period (summer months) during which these animals are refractory to FSH stimulation for augmenting oocyte collection in vivo.

In experiment 1, oocytes matured in the media containing E2 or progesterone or both steroids, with or without gonadotrophin supplementation (treatments 2–5), exhibited similar development to morulae and/or blastocysts. These responses were all significantly greater than the response in control medium with gonadotrophins but without added steroids (treatment 1, 0% blastocysts). No blastocysts were produced without the presence of one or other or both of these steroid hormones during IVM. Therefore, any effect of gonadotrophins on cumulus cells is not sufficient, and addition of steroid hormones to the culture medium is necessary to support full developmental competence of IVM oocytes. Moreover, it appears that the gonadotrophins do not augment the effects of steroid hormones on cytoplasmic maturation during IVM, because gonadotrophins did not produce any significant improvement when combined with E2 and progesterone compared with the responses obtained with steroid hormones alone. Thus, unlike the situation in vivo (Armstrong et al., 1991Go; McDonnell and Goldman, 1994Go; Graham et al., 1995Go), we did not detect any synergistic effect between the actions of E2, progesterone and gonadotrophins under in-vitro conditions. The stimulation of oocyte developmental competence in vitro by E2 and progesterone is consistent with observations in rhesus monkey and human showing a positive correlation between (i) the follicular fluid content of E2 or the progesterone to E2 ratio and (ii) embryo developmental potential and establishment of pregnancy (Botero-Ruiz et al., 1984Go; Reinthaller et al., 1987Go; Basuray et al., 1988Go; Morgan et al., 1990Go). Furthermore, E2 has been reported to act on human oocytes during IVM to enhance their fertilization and cleavage competence (Tesarik and Mendoza, 1995Go). However, the classic nuclear estrogen receptor (ER) is not expressed in rhesus monkey granulosa cells (Hild-petito et al., 1988Go; Chandrasekher et al., 1994Go), and the progesterone receptor (PR) is absent from granulosa cells until after the mid-cycle LH surge (Chandrasekher et al., 1994Go). Therefore, we propose that the actions of E2 and progesterone on primate oocyte developmental competence might be mediated rapidly through the non-genomic mechanism via cell membrane receptors, as shown for meiotic control in studies with Xenopus (Wasserman et al., 1980Go; Sadler and Maller, 1982Go; Liu and Patino, 1993Go; Bayaa et al., 2000Go). In Xenopus, progesterone operates through a plasma membrane-associated tyrosine kinase to activate phospholipase C (Morrison et al., 2000Go). It will be interesting to determine whether progesterone exerts its action on cytoplasmic maturation in mammalian oocytes via a similar mechanism.

Cell membrane ER, which transfer the signal cascade through Ca2+ release after binding with E2, have been detected in pig and chicken granulosa cells (Morley et al., 1992Go), and were also reported in human oocytes to mediate the stimulatory action of E2 on oocyte fertilization and cleavage potentials (Tesarik and Mendoza, 1995Go). This is consistent with the elevation of intracellular calcium and activation of plasma membrane tyrosine kinase by progesterone during IVM in amphibians (Wasserman et al., 1980Go; Morrison et al., 2000Go). In trilostane-treated rhesus monkeys, replacement therapy with the progestin agonist R5020 was able to restore ovulation but failed to restore the oocyte’s fertilizability and therefore indicates the alternative mechanism of progesterone action directly on cell membranes (Hibbert et al., 1996Go). However, in rhesus monkey follicles, no cell membrane ER or PR has been reported to date. Therefore, further studies are needed to clarify the mechanism of E2 and progesterone actions on primate oocyte maturation in vitro.

As in the adult monkeys, maturation of COC from prepubertal females in medium supplemented with E2 or progesterone also supported significantly higher frequencies of morula development (Table IV, treatments 2 and 3) when compared with the control (treatment 1). These results confirm the conclusion from experiment 1 that E2 and progesterone can improve oocyte developmental competence (cytoplasmic maturation). However, there was a difference between the COC from adult and prepubertal animals. The COC from prepubertal females had poorer developmental response to steroids than their counterparts from adult ovaries as assessed by the embryos’ progression to blastocysts. The presence of E2 or progesterone in media for oocyte IVM failed to stimulate blastocyst formation in prepubertal monkeys (Table IV), whereas 14–20% blastocysts were obtained from oocytes of adults after IVM with steroids (Table III). Similarly, in cattle, the marked positive effect of serum on cow oocyte maturation was not observed with calf oocytes (Lonergan et al., 1994Go; Khatir et al., 1996Go). Comparing the developmental responses between E2/progesterone-supported IVM oocytes from adult versus prepubertal monkeys (Tables III and IV), it is evident that while 31–44% of morulae progressed into blastocysts in experiment 1, only 0–7% did so in experiment 2. This discrepancy illustrates the importance of the morula-to-blastocyst transformation for revealing defects in oocytes or embryos, as well as reinforcing the difference in competence of the IVM oocytes from the young versus adult animals. It has been suggested that mammalian oocytes obtain their developmental competence in a progressive or stepwise manner during maturation, and that this process may be distorted by IVM (Eppig et al., 1994Go; Eppig, 1996Go; Schramm and Bavister, 1999Go). The ability to progress to the blastocyst stage may be acquired relatively late in oocyte growth and maturation (Eppig and Schroeder, 1989Go; Eppig et al., 1994Go; Schramm and Bavister, 1999Go). The absence of E2 and progesterone actions on blastocyst formation in experiment 2 possibly reflects that the mechanism responsible for blastocyst formation is not established in COC before puberty. However, in-vivo priming with FSH markedly improved the competence of prepubertal monkey oocytes to progress to the blastocyst stage (Zheng et al., 2001bGo). Thus, we propose that in rhesus monkeys, the in-vivo action of FSH is important for COC to respond to steroids by developing the capability to form blastocysts.

In summary, the present study demonstrates that E2 and progesterone stimulate rhesus monkey oocyte developmental capacity during IVM, and suggests an in-vivo role of these steroid hormones in local modulation of oocyte cytoplasmic maturation in primates. It shows that gonadotrophins in the absence or in the presence of steroid hormones are not effective in stimulating either nuclear or cytoplasmic maturation in vitro of monkey oocytes. Additionally, we have validated the use of a chemically defined culture system for oocytes from unstimulated rhesus monkeys that allows examination of the contribution of specific agents to IVM. This culture system can support frequencies of blastocyst formation similar to those obtained with oocytes from FSH-primed adult rhesus monkeys matured in vitro using undefined, serum-containing culture media (Zheng et al., 2001bGo). However, this system still does not support rhesus oocyte developmental competence at the levels observed with oocytes matured in vivo (Schramm and Bavister, 1999Go). We anticipate that defined culture systems like this will assist basic research into cytoplasmic maturation and perhaps can be applied to the clinical treatment of human infertility using immature oocytes collected from natural cycles.


    Acknowledgements
 
This research was supported by the major state basic research development program G2000016108, and the National Institutes of Health, USA (grant no. HD22023 and Training Grant no. RO3-TW00840 from the Fogarty International Center).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Abeydeera, L.R., Wang, W.H., Cantley, T.C., Riecke, A., Murphy, C.N., Prather, R.S. and Day, B.N. (2000) Development and viability of pig oocytes matured in a protein-free medium containing epidermal growth factor. Theriogenology, 54, 787–797.[CrossRef][ISI][Medline]

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Basuray, R., Rawlins, R.G., Radwanska, E., Henig, I., Sachdeva, S., Tummon, I., Binor, Z., and Dmowski, W.P. (1988) High progesterone/estradiol ratio in follicular fluid at oocyte aspiration for in vitro fertilization as a predictor of possible pregnancy. Fertil. Steril., 49, 1007–1011.[ISI][Medline]

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Submitted on April 17, 2001; resubmitted on May 22, 2003; accepted on June 26, 2003.