Role of estradiol-17 on nuclear and cytoplasmic maturation of pig oocytes

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1 Animal Reproduction Science 78 (2003) Role of estradiol-17 on nuclear and cytoplasmic maturation of pig oocytes M.A.N. Dode a,, C.N. Graves b a Animal Reproduction Laboratory, Embrapa Genetic Resources and Biotechnology, Parque Estação Biológica, Avenue W5 Final Norte, Brasília CEP , DF, Brazil b Department of Animal Science, University of Illinois, 1207 West Gregory Drive, Urbana, IL 61801, USA Received 3 September 2002; received in revised form 21 January 2003; accepted 6 March 2003 Abstract The role of estradiol-17 on nuclear and cytoplasmic maturation of pig oocytes was investigated in the present study. To determine the estradiol effect, oocytes were cultured for 42 h in a steroid free medium composed of mtcm-199 supplemented with LH, FSH and 10% charcoal extracted follicular fluid. Estradiol receptor (ER), detected by a binding assay, were present in cumulus cells and oocytes during maturation with higher levels observed at 24 h of culture in the oocytes and at 36 h in the cumulus cells. To block estradiol action an antiestrogen (1-p-dimethylaminoethoxyphenyl-1,2- diphenyl-1-butene (tamoxifen)) was added to the maturation medium at various concentrations. The percentage of treated oocytes that underwent nuclear maturation was similar (P >0.05) to the control group. Cytoplasmic maturation, determined by the ability to form female pronucleus (FPN) and male pronucleus (MPN), was not different (P >0.05) among all groups. The presence of 4-hydroxy-4-androstene-3-17-dione (4-OHA) also did not influence nuclear (P >0.05) or cytoplasmic maturation (P >0.05). The results suggest that estradiol is not involved in maturation of pig oocytes. However, the present experiment used pronuclei formation as the endpoint, no studies were done in regard to estradiol s effects on the embryonic development Elsevier Science B.V. All rights reserved. Keywords: Oocyte maturation; Porcine; Oestrogen 1. Introduction The presence of steroids in the follicular fluid before and during maturation (McNatty, 1978; Ainsworth et al., 1980) suggests that they may play a role in oocyte maturation. In fact, it has been shown that estradiol as well as other steroids are involved in keeping the oocytes Corresponding author. Tel.: ; fax: address: margot@cenargen.embrapa.br (M.A.N. Dode) /03/$ see front matter 2003 Elsevier Science B.V. All rights reserved. doi: /s (03)

2 100 M.A.N. Dode, C.N. Graves / Animal Reproduction Science 78 (2003) in meiotic arrest (McGaughey, 1977; Richter and McGaughey, 1979; Smith and Tenney, 1980; Racowsky and McGaughey, 1982; Kaji et al., 1987; Barrett and Powers, 1993; Mingoti et al., 1995). It also has been suggested that estradiol is important in the oocyte acquisition of fertilization competence (Yoshimura et al., 1987; Younis et al., 1989; Sirotkin, 1992; Pellicer, 1997). However, the specific role of estrogens in follicular and oocyte maturation, ovulation and embryo development seems to be species dependent (Moudgal et al., 1996) and is currently unknown in various species. In a previous experiment we have shown that pig cumulus oocyte complexes (COCs) secrete estradiol during culture in a steroid free medium, probably as a consequence of the action of gonadotropins (Dode and Graves, 2002). Secretion of estradiol by human and bovine cumulus cells has also been reported (Chian et al., 1999; Mingoti et al., 2002). These findings support the idea that estradiol may exert an effect during oocyte maturation, which can be indirectly via cumulus cells or directly in the oocyte. If estradiol is mediating changes at the cellular level, that result in the oocyte acquiring the ability to be fertilized, the presence of estradiol receptor (ER) is essential in mediating those changes. In humans the presence of mrna for ER was observed in oocytes and COCs but not in cumulus cells (Wu et al., 1993), whereas in the sheep ER and its mrna was observed only in the cumulus cells surrounding immature oocytes in small (less than 2 mm) and medium size (2 4 mm) follicles (Tomanek et al., 1997). However, the presence of estradiol receptors during the maturation period has not been reported. Because secretion of estradiol by the COCs occurs during maturation it is difficult to determine the estradiol effects unless the action or the secretion of estradiol is blocked. Non-steroidal antiestrogen compounds have been shown to antagonize many of the actions of estrogen and have been widely used to inactivate ER in human cancer cells (Nardulli and Katzenellenbogen, 1986; Read et al., 1988; Katzenellenbogen and Norman, 1990; Aronica and Katzenellenbogen, 1991; Aronica, 1993). Tamoxifen is a very common antiestrogen that competes with estrogen for the ER and forms a tamoxifen receptor complex, which is inactive in transcription (Horwitz and McGuire, 1978; Clark and Markaverich, 1988). Although tamoxifen has been shown to be a mixed agonist antagonist of estradiol action (Clark and Markaverich, 1988; Clark et al., 1992), when used in high concentrations (10 7 to 10 6 M) the agonist effect is eliminated because it fully inhibits estradiol, being a complete antagonist (Katzenellenbogen et al., 1987). Besides estrogen action by classical receptor-mediated pathway, several lines of evidence suggested that estradiol can also act directly in the oocytes by producing changes in the reactivity of its Ca 2+ release mechanism supposed to be involved in oocyte cytoplasmic maturation (Tesarik and Mendoza, 1995, 1997; Chian et al., 1999). Therefore, the steroidal effect in cells can be by two different modes of action: the classical and the non-genomic which does not involve a modification of gene activity. Based in this evidence another approach has to be used in order to study the influence of estradiol, which could be to block its synthesis by using enzyme inhibitors of the steroidogenesis pathway. Aromatase is the steroidogenic enzyme responsible for catalyzing the conversion of androgens to estrogens. A synthetic androgenic compound 4-hydroxy-4-androstene-3-17-dione (4-OHA), which binds irreversible to the aromatase enzyme (Brodie et al., 1977; Wozniak et al., 1992; Yu et al., 1993), has been shown to be an efficient inhibitor of the aromatization of 4-androstene-3-17-dione to estrone and estradiol in vitro and in vivo (Brodie et al.,

3 M.A.N. Dode, C.N. Graves / Animal Reproduction Science 78 (2003) , 1981a,b; Brodie and Longcope, 1989). Because COCs secrete estradiol during culture and that estradiol can act in the oocyte without crossing its plasma membrane, by its non-genomic action, the use of a specific and potent aromatase inhibitor contributes to the investigation of the role of estradiol in oocyte maturation. The objectives of the present study were to examine the presence of receptors for estradiol during maturation and to determine the role of estradiol during maturation by blocking the estradiol action using an antiestrogen agent (tamoxifen), and by blocking estradiol secretion using an aromatase inhibitor (4-OHA). 2. Materials and methods 2.1. Determination of presence of estrogen receptors in the cumulus cells and oocytes The presence of estradiol receptors in the pig oocyte and cumulus cells before and after maturation was determined by a receptor binding assay. The affinity of sex steroids for their receptors in the pig has been previously determined (Stanchev et al., 1990). Six hundred COCs were used, with 300 COCs used for each assay. MCF-7 human breast cancer cells, which are ER-positive cells, and 231N human breast cancer cells, which are low ER cells, were used as control cells. The COCs were collected from medium size follicles (2 5 mm) as described previously (Dode, 1994). Half of the COCs was used for the before maturation analysis and the other half was cultured for 42 h at 38.5 C in mtcm-199 supplemented with FSH, LH and 10% pig follicular fluid (pff). The preparation of the cells was similar for both the before and the after maturation groups. For cell preparation, COCs were denuded from their cumulus cells and then used for the estradiol receptor assay. After separation, the oocytes and the cumulus cells were centrifuged at 300 g for 5 min. Cell pellets were then homogenized using 200 l of extraction buffer TEG (50 mm Tris HCL, 7.5 mm EDTA, 10% glycerol, 0.6 M NaCl, ph 7.4), and immediately frozen in dry ice and stored at 80 C. Immediately after thawing, 10 l of protease inhibitors (1 mm phenylmethylsulfonylfluoride, 0.1 mg/ml soybean trypsin inhibitor and 0.1 mg/ml leupeptin) were added and the cells were homogenized. The extracts then were vortexed every 5 min for 15 min. Each extract was transferred into two (for duplicates) ultracentrifuge tubes and 200 l of5 TEG buffer were added. Aliquots of each extract were used for protein concentration determinations by the BCA method (Aronica, 1993). Extracts were then centrifuged at 130,000 g for 25 min. For the ER assay, aliquots of the cell extract supernatant were incubated with 10 nm [ 3 H]estradiol in the absence and presence of 1000 nm (100-fold excess) unlabeled estradiol for 15 h at 4 C to measure total and non-specific binding, respectively. After incubation free steroids were removed by treatment with charcoal dextran for 7 min. After separation of the charcoal, by 6 min centrifugation (12,000 g), 50 l of the supernatant were transferred to a vial, 5 ml of scintillation fluid were added and the contents assayed for radioactivity in a scintillation counter. Specific binding (total minus non-specific binding) values are expressed in fmol/mg of protein.

4 102 M.A.N. Dode, C.N. Graves / Animal Reproduction Science 78 (2003) Determination of estrogen receptors in the cumulus cells and oocytes at various time points during maturation One thousand COCs were collected from 2 to 5 mm diameter follicles and cultured for 0, 24, 36 or 42 h at 38.5 C, in mtcm-199 supplemented with FSH, LH and 10% pff. At each time point, 250 COCs were denuded and the cumulus cells and oocytes were used for a ER binding assay. Although the procedure was the same as described above, for this assay T47D cells, which contain low levels of ER (Read et al., 1988), were used as control cells. The results are expressed in fmol of receptor/mg of protein Action of estradiol antagonist on in vitro maturation of pig oocytes In the present study, an antiestrogen agent was tested in different concentrations in a three replicate experiment. Oocytes obtained from 2 to 5 mm diameter follicles were matured in the presence of 0, 10 8,10 7,10 6 or 10 5 M1-p-dimethylaminoethoxyphenyl-1,2- diphenyl-1-butene (tamoxifen; Sigma) in mtcm-199 supplemented with LH, FSH and 10% charcoal extracted follicular fluid. After 42 h of culture, the oocytes were fertilized in vitro and stained to verify nuclear and cytoplasmic maturation. The charcoal extracted follicular fluid showed, by RIA, undetectable concentrations (less than 5 pg) of progesterone, estradiol and testosterone. Stock solutions of the various concentrations of tamoxifen were made by dissolving the tamoxifen in absolute ethanol. The appropriate volume was added in such a way that the final concentration of ethanol never exceeded 0.2% of the volume of the incubation medium. An equivalent volume of ethanol was added to the medium that did not contain the drug. The fertilization medium contained TCM-199, d-glucose (0.55 mg/ml), Ca-lactate (0.90 mg/ml), Na-pyruvate (0.10 mg/ml), streptomycin (0.05 mg/ml), penicillin (100 IU/ml), 10% FCS and caffeine (0.40 mg/ml). For the IVF spermatozoa in a final concentration of cells/ml were co-incubated with the in vitro matured oocytes. After 8 h of culture, the oocytes were transferred to a drop of BMOC-2 medium and culture for an additional 12 h. At the end of incubation period, the oocytes were fixed with acetic:alcohol (1:3) and stained with lacmoid to identify presence of spermatozoa, sperm head decondensation and pronucleus formation. The presence of unswollen or swollen sperm heads in the ooplasma of matured oocytes was considered as an indicator of sperm penetration. The presence of two or more pronuclei, with the second polar body in the perivitelline space and detached sperm tail in the ooplasma, was used as a criterion for male pronucleus (MPN) formation Effect of the inhibition of estradiol-17β production on in vitro maturation of pig oocytes Estradiol-17 synthesis by COCs was inhibited at the step of the conversion of androgens to estradiol by using an aromatase inhibitor, 4-OHA, 1 at a concentration of 10 6 M. 1 The aromatase inhibitor was generously provided by Dr. Brodie, School of Medicine, University of Maryland, Baltimore.

5 M.A.N. Dode, C.N. Graves / Animal Reproduction Science 78 (2003) Stock solutions of the aromatase inhibitor and estradiol-17 were made by dissolving them in absolute ethanol and stored at 4 C. The dilutions were made to added to the medium a total concentration of 0.2% of ethanol. A similar volume of ethanol was added to the medium in which the compounds were not added. In the present experiment, the oocytes were matured in mtcm-199 in the presence of LH and FSH, supplemented with different components according to each treatment. The medium was supplemented with either pff (treatment 1), or pff and 10 6 M aromatase inhibitor (treatment 2), or charcoal extracted pff (treatment 3), or charcoal extracted pff and 10 6 M aromatase inhibitor (treatment 4) or charcoal extracted pff, 10 6 M aromatase inhibitor and 11 M of estradiol-17 (treatment 5). All incubations were conducted for 42 h at 38.5 C. Following maturation, the oocytes were fertilized in vitro, fixed and stained Statistical analysis The data expressed as percentages were transformed by arcsine-transformation. For each experiment, an analysis of variance was performed using a SAS statistical program (SAS Institute Inc., 2003). The treatments were compared by a randomized complete block design and the difference among means by a t-test. 3. Results Data for the concentrations of ER, determined by ligand binding before and after maturation, are included in Table 1. Before maturation, both cumulus cells and oocytes showed the presence of ER when compared with the control cells. After maturation, the cumulus cells showed a 2.6-fold increase in ER content whereas in the oocytes the concentrations remained similar to the pre-incubation level. When the amount of ER was measured at various time points during maturation (Table 2), the greatest concentration of ER in cumulus cells was observed at 36 h of maturation. In the oocytes a seven-fold increase in ER concentrations was observed in the first 24 h of maturation. Nuclear maturation was not influenced by the presence of any concentration of tamoxifen (Table 3) due to the similar percentage of oocytes that were arrested at the germinal vesicle stage (P = ) and that reached the metaphase II stage (P = ). The analysis of Table 1 Estrogen receptor content in cumulus cells and oocytes before and after in vitro maturation Cells Estrogen receptor (fmol/mg of protein) MCF N 12.0 CC a before maturation 69.0 CC a after maturation Oocytes before maturation 41.0 Oocytes after maturation 55.0 a Cumulus cells stripped from the oocytes.

6 104 M.A.N. Dode, C.N. Graves / Animal Reproduction Science 78 (2003) Table 2 Estrogen receptor content in the cumulus cells and oocytes at various time points during in vitro maturation Cells Concentration of estrogen receptor (fmol/mg of protein) 0 h 24 h 36 h 42 h Cumulus cells Oocytes T47D 47.2 variance showed no effect of treatment in the proportion of oocytes penetrated (P = ). A significant difference was observed when the means were compared, however, the differences did not occur in a dose response manner, with the greater percentage of penetration occurring when M of the antiestrogen was present. The percentage of polyspermic oocytes was not different among the treatment groups (P = ). Similarly, the ability of the oocyte to de-condense the sperm head and to form MPN was not influenced by the presence of tamoxifen (P = and P = , respectively). However, the percentage of oocytes having MPN tended to be less in the groups in which tamoxifen was present, while the addition of estradiol raised the percentage to values similar to the control group. When oocytes were matured in the presence of an aromatase inhibitor, the proportion of oocytes that underwent nuclear maturation (P = ) was similar to the control groups (Table 4). In addition, no difference was observed in the proportion of oocytes penetrated, polyspermic, showing decondensing sperm heads and MPN formation when treated groups were compared with the control (P = ). 4. Discussion Due to the large number of oocytes needed for each assay a Scatchard plot analysis, to determine the binding affinity (K d ) of these receptors for estradiol, was not done. For this analysis the K d was based on that reported for other reproductive tissues in the pig, because it has been reported that ER in mouse uterine cells (Aronica, 1993), rat hypothalamus, pituitary and uterus (Kelner et al., 1982), porcine oviduct cells (Stanchev et al., 1985), porcine cervix cells (Stanchev et al., 1984) and porcine uterine cells (Stanchev et al., 1990) have comparable values. In addition, an immunoblotting analysis that would confirm the presence of ER protein, was not done. However, the presence of estradiol receptors in cumulus cells and oocytes, was indicated by the receptor biding assay. The results indicated that estrogen receptors are present in the cumulus cells as well as in the oocytes at the time of maturation. The intracellular concentration of steroid receptors is an important factor in determining the responsive state of the target cells. The marked increase in the ER content in the oocytes at 24 h of culture and in the cumulus cells at 36 h suggest that oocytes and cumulus cells become more responsive to estradiol present at or secreted during the time of maturation. What causes the increase in ER content during maturation is not known, although it is well known that steroid receptors are influenced by endogenous and exogenous steroids and growth factors (Clark and Markaverich, 1988; Read et al., 1989) in various target tissues. An increase in

7 Table 3 Effect of presence of various concentration of tamoxifen on in vitro maturation of pigs oocytes Tamoxifen concentration (M) Oocyte (no.) MII a oocytes, GV b oocytes, Oocytes penetrated, Oocytes polyspermics, No. of sperms/oocytes Oocytes DSH c, Oocytes PN d, ± 0.02 a 3.5 ± 0.99 a 45.9 ± 0.94 abc 60.5 ± a 2.2 ± 0.01 a 54.8 ± 0.06 a 45.1 ± 0.06 a ± 0.73 a 14.0 ± 0.50 a 34.2 ± 0.16 c 29.1 ± 2.00 a 2.3 ± 0.01 a 56.5 ± 2.15 a 27.3 ± 0.43 a ± 0.16 a 18.0 ± 0.16 a 63.0 ± 1.10 ab 63.6 ± 2.00 a 2.3 ± 0.01 a 56.6 ± 1.10 a 25.1 ± 0.13 a ± 0.32 a 4.1 ± 1.20 a 41.0 ± 0.11 bc 53.2 ± 0.52 a 2.4 ± 0.01 a 53.8 ± 0.74 a 29.0 ± 0.07 a ± 1.80 a 6.8 ± 1.8 a 65.3 ± 0.63 a 60.8 ± 1.8 a 2.5 ± 0.01 a 43.4 ± 0.52 a 25.6 ± 0.83 a Estradiol ± 0.10 a 10.6 ± 0.03 a 48.7 ± 0.99 abc 40.1 ± 2.40 a 2.3 ± 0.01 a 53.3 ± 1.00 a 44.9 ± 2.10 a Within each column values with different letters (a c) are significantly different (P <0.05). a Oocytes at metaphase II stage after in vitro maturation. b Oocytes at germinal vesicle stage after in vitro maturation. c Oocytes containing decondensed sperm head after in vitro fertilization. d Oocytes containing one female pronucleus and at least one male pronucleus after in vitro fertilization. M.A.N. Dode, C.N. Graves / Animal Reproduction Science 78 (2003)

8 Table 4 Effect of an aromatase inhibitor (4-HOA) in the culture medium on in vitro maturation of pigs oocytes Treatment Oocyte (no.) MII a oocytes, GV b oocytes, Oocytes penetrated, Oocytes polyspermics, No. of sperms/oocytes Oocytes DSH c, Oocytes PN d, Follicular fluid ± ± ± ± ± ± ± 0.74 Follicular fluid + I e ± ± ± ± ± ± ± 0.47 CE follicular fluid f ± ± ± ± ± ± ± 0.27 CE follicular fluid f + I e ± ± ± ± ± ± ± 0.66 CE follicular fluid f + I e + estradiol g ± ± ± ± ± ± ± 0.32 a Oocytes at metaphase II stage after in vitro maturation. b Oocytes at germinal vesicle stage after in vitro maturation. c Oocytes containing decondensed sperm head after in vitro fertilization. d Oocytes containing one female pronucleus and at least one male pronucleus after in vitro fertilization. e Aromatase inhibitor (4-hydroxy-4-androstene-3-17-dione) added to the maturation medium at a concentration of M. f Charcoal extracted porcine follicular fluid added to the maturation medium at concentration of 10%. g Estradiol 17- added to the maturation medium at concentration of 11 M. 106 M.A.N. Dode, C.N. Graves / Animal Reproduction Science 78 (2003)

9 M.A.N. Dode, C.N. Graves / Animal Reproduction Science 78 (2003) camp has been reported to induce a significant increase in intracellular ER (Aronica, 1993). If this is true for other target cells it is possible that LH, which acts by elevating camp concentration (Dekel, 1988) could induce the ER in oocytes and cumulus cells. In addition, it has been shown that cells containing small numbers of ER when exposed to estradiol increase ER mrna and ER protein (Read et al., 1989). Because estradiol is present and secreted during maturation, it is possible that this mechanism is part of the regulation of ER numbers. However, it is important to consider that most of these observations are based on studies with uterine cells or cancer cell lines. It would be interesting to study which factor or factors are responsible for the induction of ER in the oocytes. The mechanism(s) by which ER increased earlier in the oocytes than in the cumulus cells still remains to be established. Nevertheless, studies with different cancer cells lines showed that the direction and magnitude of ER changes depend on the cell line (Read et al., 1989). The different sensitivity among various target tissues provides the mechanism by which it is possible to regulate differentially the steroid response. If a target cell at some point is less sensitive to estradiol than others it is possible to activate only a subset of steroid target tissue and not all tissues with ER. It is possible that during the first 24 h of maturation of the oocytes is a crucial time in which protein synthesis has to occur in order for GVBD to occur and for meiosis to proceed normally. The first 24 h of maturation in the pig oocyte is the time in which G2 to M phase transition occurs, with this transition being induced by the activation of maturation promoting factor (MPF) (Naito and Toyoda, 1991). The conversion of the inactive MPF involved a change in the phosphorylation and its association with some protein (cyclins) (Cran and Moor, 1990). In contrast to rodent oocytes, in which protein synthesis is not required for entry into M phase (Jagiello, 1969), the pig oocyte requires protein synthesis to undergo meiotic maturation (Moor, 1988). The possible effect of estradiol on the synthesis of proteins that is vital for the cell cycle progression was evaluated in the present study by maturating oocytes in estradiol-free medium. When oocytes were cultured in presence of various concentrations of an antiestrogen, no effect was observed in the percentage of oocytes that remained in germinal vesicle stage or that reached metaphase II stage. Similar results were obtained when an aromatase inhibitor was added to the maturation medium, which suggests that estradiol is not involved in the protein synthesis required for the MPF activity. This finding is supported by studies with ovine oocytes (Sun and Moor, 1991). Such studies showed that the nuclei of ovine oocytes could be removed at various times before GVBD without preventing the changes in protein synthesis, concluding that the presence of the nucleus is not essential for the protein synthesis before GVBD. The non-genomic effect of estradiol, however, was also evaluated in the present study by inhibiting the estradiol secretion. Estradiol has been reported to influence cytoplasmic maturation by acting at the oocyte surface (Tesarik and Mendoza, 1997) in a non-genomic signaling manner. In human oocytes, estradiol induced a rapid increase in free intracellular Ca 2+ concentration followed by a series of Ca 2+ oscillations enhancing the cytoplasmic maturation (Tesarik and Mendoza, 1995). However, the same effect was not observed in the present study, in which the absence of estradiol had no effect on cytoplasmic maturation. The male pronucleus growth factor (MPGF) develops during maturation and is essential for the formation of MPN. The control of MPGF, as well as the proteins involved in its activation, have not been established. Nevertheless, the results of some studies suggest the

10 108 M.A.N. Dode, C.N. Graves / Animal Reproduction Science 78 (2003) requirement of steroid hormones for the MPN formation (Mattioli et al., 1988; Sirotkin, 1992; Yoshimura et al., 1987). In the present study, the absence of estradiol had no significant effect either on nuclear and cytoplasmic maturation or on the other characteristics studied. The similar results obtained in the tamoxifen and aromatase inhibitor studies suggest that estradiol is not involved in MPN formation. Some consideration, however, should be given in regard to the results obtained. The presence of tamoxifen tended to effect MPN formation, which was reversed by the addition of estradiol. Whether the amounts used were effective in blocking estradiol action is not known, however, it was expected, by studies with other cell types to be sufficient to compete with estradiol and bind with its receptors. Greater concentrations could not be used because when 10 4 M of tamoxifen was tested in a preliminary study (data not shown) a detrimental effect on oocyte maturation occurred because all the oocytes were held at the germinal vesicle stage and the addition of estradiol did not reverse this effect. A similar tendency was not observed in the aromatase inhibitor experiment. It is possible that the amount of the aromatase inhibitor used in the present study was not sufficient to inhibit all the estradiol production. No measurement was made of the estradiol concentration in the medium to determine the efficacy, however, the concentration of 4-HOA used (10 6 M) should have inhibited more than 90% of the estradiol production (Brodie, personal communication). We also observed that when either the inhibitor or the antiestrogen were present some oocytes were not activated or remained at metaphase II and did not form a female pronucleus (FPN), although sperm penetration occurred and the sperm head decondensed. Further studies are needed to verify the involvement of estradiol in the ability of the oocytes to be activated. The results of the present experiment strongly suggest that estradiol is not involved in nuclear or cytoplasmic maturation of pig oocytes. Maybe the high concentration of the estradiol in the follicular fluid is only needed to keep the oocyte in meiotic arrest and it is not required for maturation itself. It is important to consider also that these results do not eliminate the effect of estradiol in cleavage and embryonic development as well as the involvement of other steroid hormones in maturation. Clarification of these aspects will require further studies. Acknowledgements This project was partially funded by the University of Illinois Agriculture Experiment Station, Grant #ILLU The authors thank the USDA Animal Program for the FSH and LH, Dr. Brodie, University of Maryland (Baltimore), for the aromatase inhibitor and Exel Corp., Beardstown, IL, for furnishing the ovaries used in this study. References Ainsworth, L., Tsang, B.K., Downey, B.R., Marcus, G.J., Armstrong, D.T., Interrelationship between follicular fluid steroid levels, gonadotrophic stimuli and oocyte maturation during preovulatory development of porcine follicles. Biol. Reprod. 23,

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