Animal Reproduction Science 78 (2003) 99–110 Role of estradiol-17␤ on nuclear and cytoplasmic a Animal Reproduction Laboratory, Embrapa Genetic Resources and Biotechnology, Parque Estação Biológica, Avenue W5 Final Norte, Bras´ılia CEP 70770-900, 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 freemedium composed of mTCM-199 supplemented with LH, FSH and 10% charcoal extracted follic-ular fluid. Estradiol receptor (ER), detected by a binding assay, were present in cumulus cells andoocytes during maturation with higher levels observed at 24 h of culture in the oocytes and at 36 h inthe 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) tothe 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 presenceof 4-hydroxy-4-androstene-3-17-dione (4-OHA) also did not influence nuclear (P > 0.05) or cyto-plasmic maturation (P > 0.05). The results suggest that estradiol is not involved in maturation ofpig oocytes. However, the present experiment used pronuclei formation as the endpoint, no studieswere done in regard to estradiol’s effects on the embryonic development.
2003 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 ( 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.: +55-61-448-4659; fax: +55-61-340-3658.
E-mail address: (M.A.N. Dode).
0378-4320/03/$ – see front matter 2003 Elsevier Science B.V. All rights reserved.
doi:10.1016/S0378-4320(03)00080-0 M.A.N. Dode, C.N. Graves / Animal Reproduction Science 78 (2003) 99–110 in meiotic arrest (It also has been suggested that estradiol is important in the oocyte acquisitionof fertilization competence However, the specific role of estrogens in follicular and oocyte maturation,ovulation and embryo development seems to be species dependent 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 ofthe action of gonadotropins Secretion of estradiol by human andbovine cumulus cells has also been reported (findings support the idea that estradiol may exert an effect during oocyte maturation, whichcan 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 mediatingthose changes. In humans the presence of mRNA for ER was observed in oocytes andCOCs but not in cumulus cells whereas in the sheep ER and its mRNAwas observed only in the cumulus cells surrounding immature oocytes in small (less than2 mm) and medium size (2–4 mm) follicles (However, the presenceof 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 actionsof estrogen and have been widely used to inactivate ER in human cancer cells (Tamoxifen is a very common antiestrogenthat competes with estrogen for the ER and forms a tamoxifen–receptor complex, whichis inactive in transcription Although tamoxifen has been shown to be a mixed agonist–antagonist of estradiol actionused in high concentrations (10−7 to10−6 M) the agonist effect is eliminated because it fully inhibits estradiol, being a completeantagonist ( 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 thereactivity of its Ca2+ release mechanism supposed to be involved in oocyte cytoplasmicmaturation (Therefore, the steroidaleffect in cells can be by two different modes of action: the classical and the non-genomicwhich does not involve a modification of gene activity. Based in this evidence anotherapproach has to be used in order to study the influence of estradiol, which could be toblock its synthesis by using enzyme inhibitors of the steroidogenesis pathway. Aromataseis the steroidogenic enzyme responsible for catalyzing the conversion of androgens to es-trogens. A synthetic androgenic compound 4-hydroxy-4-androstene-3-17-dione (4-OHA),which binds irreversible to the aromatase enzyme has been shown to be an efficient inhibitor of the aromatizationof 4-androstene-3-17-dione to estrone and estradiol in vitro and in vivo ( M.A.N. Dode, C.N. Graves / Animal Reproduction Science 78 (2003) 99–110 Because COCs secrete estradiol during cul-ture and that estradiol can act in the oocyte without crossing its plasma membrane, by itsnon-genomic action, the use of a specific and potent aromatase inhibitor contributes to theinvestigation 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 theestradiol action using an antiestrogen agent (tamoxifen), and by blocking estradiol secretionusing an aromatase inhibitor (4-OHA).
2. Materials and methods
2.1. Determination of presence of estrogen receptors in the cumuluscells 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 theirreceptors in the pig has been previously determined ( 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 arelow ER cells, were used as control cells.
The COCs were collected from medium size follicles (2–5 mm) as described previously (Half of the COCs was used for the before maturation analysis and the otherhalf 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 andthe after maturation groups. For cell preparation, COCs were denuded from their cumuluscells and then used for the estradiol receptor assay. After separation, the oocytes and thecumulus cells were centrifuged at 300 × g for 5 min. Cell pellets were then homogenizedusing 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 phenylmethylsulfonylflu- oride, 0.1 mg/ml soybean trypsin inhibitor and 0.1 mg/ml leupeptin) were added and thecells were homogenized. The extracts then were vortexed every 5 min for 15 min. Each ex-tract was transferred into two (for duplicates) ultracentrifuge tubes and 200 ␮l of 5× TEGbuffer were added. Aliquots of each extract were used for protein concentration determina-tions by the BCA method (Extracts were then centrifuged at 130,000 × gfor 25 min.
For the ER assay, aliquots of the cell extract supernatant were incubated with 10 nM [3H]estradiol in the absence and presence of 1000 nM (100-fold excess) unlabeled estradiolfor 15 h at 4 ◦C to measure total and non-specific binding, respectively. After incubation freesteroids were removed by treatment with charcoal dextran for 7 min. After separation of thecharcoal, by 6 min centrifugation (12,000 × g), 50 ␮l of the supernatant were transferredto a vial, 5 ml of scintillation fluid were added and the contents assayed for radioactivityin a scintillation counter. Specific binding (total minus non-specific binding) values areexpressed in fmol/mg of protein.
M.A.N. Dode, C.N. Graves / Animal Reproduction Science 78 (2003) 99–110 2.2. Determination of estrogen receptors in the cumulus cells and oocytesat 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. Ateach time point, 250 COCs were denuded and the cumulus cells and oocytes were used fora ER binding assay. Although the procedure was the same as described above, for this assayT47D cells, which contain low levels of ER (were used as control cells.
The results are expressed in fmol of receptor/mg of protein.
2.3. 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 maturedin the presence of 0, 10−8, 10−7, 10−6 or 10−5 M 1-p-dimethylaminoethoxyphenyl-1,2-diphenyl-1-butene (tamoxifen; Sigma) in mTCM-199 supplemented with LH, FSH and10% charcoal extracted follicular fluid. After 42 h of culture, the oocytes were fertilized invitro 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 thefinal 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 of5 × 105 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 additional12 h. At the end of incubation period, the oocytes were fixed with acetic:alcohol (1:3) andstained with lacmoid to identify presence of spermatozoa, sperm head decondensation andpronucleus formation. The presence of unswollen or swollen sperm heads in the ooplasmaof matured oocytes was considered as an indicator of sperm penetration. The presence oftwo or more pronuclei, with the second polar body in the perivitelline space and detachedsperm tail in the ooplasma, was used as a criterion for male pronucleus (MPN) formation.
2.4. Effect of the inhibition of estradiol-17β production on in vitro maturationof 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,at a concentration of 10−6 M.
1 The aromatase inhibitor was generously provided by Dr. Brodie, School of Medicine, University of Maryland, M.A.N. Dode, C.N. Graves / Animal Reproduction Science 78 (2003) 99–110 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 themedium a total concentration of 0.2% of ethanol. A similar volume of ethanol was addedto 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. Themedium was supplemented with either pFF (treatment 1), or pFF and 10−6 M aromataseinhibitor (treatment 2), or charcoal extracted pFF (treatment 3), or charcoal extracted pFFand 10−6 M aromatase inhibitor (treatment 4) or charcoal extracted pFF, 10−6 M aromataseinhibitor and 11 ␮M of estradiol-17␤ (treatment 5). All incubations were conducted for 42 hat 38.5 ◦C. Following maturation, the oocytes were fertilized in vitro, fixed and stained.
The data expressed as percentages were transformed by arcsine-transformation. For each experiment, an analysis of variance was performed using a SAS statistical program (The treatments were compared by a randomized complete block designand the difference among means by a t-test.
3. Results
Data for the concentrations of ER, determined by ligand binding before and after matu- ration, are included in maturation, both cumulus cells and oocytes showedthe presence of ER when compared with the control cells. After maturation, the cumuluscells showed a 2.6-fold increase in ER content whereas in the oocytes the concentrationsremained similar to the pre-incubation level.
When the amount of ER was measured at various time points during maturation ( the greatest concentration of ER in cumulus cells was observed at 36 h of maturation. Inthe oocytes a seven-fold increase in ER concentrations was observed in the first 24 h ofmaturation.
Nuclear maturation was not influenced by the presence of any concentration of tamoxifen (due to the similar percentage of oocytes that were arrested at the germinal vesiclestage (P = 0.5288) and that reached the metaphase II stage (P = 0.4575). The analysis of Table 1Estrogen receptor content in cumulus cells and oocytes before and after in vitro maturation a Cumulus cells stripped from the oocytes.
M.A.N. Dode, C.N. Graves / Animal Reproduction Science 78 (2003) 99–110 Table 2Estrogen receptor content in the cumulus cells and oocytes at various time points during in vitro maturation Concentration of estrogen receptor (fmol/mg of protein) variance showed no effect of treatment in the proportion of oocytes penetrated (P = 0.0745).
A significant difference was observed when the means were compared, however, the differ-ences did not occur in a dose response manner, with the greater percentage of penetrationoccurring when 1 × 10−5 M of the antiestrogen was present. The percentage of polyspermicoocytes was not different among the treatment groups (P = 0.8913). Similarly, the abilityof the oocyte to de-condense the sperm head and to form MPN was not influenced by thepresence of tamoxifen (P = 0.9381 and P = 0.3218, respectively). However, the percent-age 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 = 0.5407) was similar to the control groupsIn addition, no difference was observed in the proportion of oocytes penetrated,polyspermic, showing decondensing sperm heads and MPN formation when treated groupswere compared with the control (P = 0.3403).
4. Discussion
Due to the large number of oocytes needed for each assay a Scatchard plot analysis, to determine the binding affinity (Kd) of these receptors for estradiol, was not done. For thisanalysis the Kd was based on that reported for other reproductive tissues in the pig, becauseit has been reported that ER in mouse uterine cells (rat hypothalamus,pituitary and uterus (porcine oviduct cells porcine cervix cells (porcine uterine cells (have comparable values. In addition, an immunoblotting analysis that would confirm thepresence of ER protein, was not done. However, the presence of estradiol receptors incumulus cells and oocytes, was indicated by the receptor biding assay. The results indicatedthat estrogen receptors are present in the cumulus cells as well as in the oocytes at the timeof 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 at24 h of culture and in the cumulus cells at 36 h suggest that oocytes and cumulus cells becomemore responsive to estradiol present at or secreted during the time of maturation. What causesthe increase in ER content during maturation is not known, although it is well known thatsteroid receptors are influenced by endogenous and exogenous steroids and growth factorsin various target tissues. An increase in Table 3Effect of presence of various concentration of tamoxifen on in vitro maturation of pigs oocytes 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.
Effect of an aromatase inhibitor (4-HOA) in the culture medium on in vitro maturation of pigs oocytes Oocytes penetrated, Oocytes polyspermics, percentage ± S.E. percentage ± S.E. percentage ± S.E.
sperms/oocytes percentage ± S.E. percentage ± S.E.
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 1 × 10−6 M.
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.
M.A.N. Dode, C.N. Graves / Animal Reproduction Science 78 (2003) 99–110 cAMP has been reported to induce a significant increase in intracellular ER (If this is true for other target cells it is possible that LH, which acts by elevating cAMPconcentration induce the ER in oocytes and cumulus cells. In addition,it has been shown that cells containing small numbers of ER when exposed to estradiolincrease ER mRNA and ER protein (Because estradiol is present andsecreted during maturation, it is possible that this mechanism is part of the regulation of ERnumbers. However, it is important to consider that most of these observations are based onstudies with uterine cells or cancer cell lines. It would be interesting to study which factoror 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 showedthat the direction and magnitude of ER changes depend on the cell line (The different sensitivity among various target tissues provides the mechanism by which itis possible to regulate differentially the steroid response. If a target cell at some point isless sensitive to estradiol than others it is possible to activate only a subset of steroid targettissue and not all tissues with ER. It is possible that during the first 24 h of maturation ofthe oocytes is a crucial time in which protein synthesis has to occur in order for GVBD tooccur and for meiosis to proceed normally. The first 24 h of maturation in the pig oocyte isthe time in which G2 to M phase transition occurs, with this transition being induced by theactivation of maturation promoting factor (MPF) (The conversionof the inactive MPF involved a change in the phosphorylation and its association with someprotein (cyclins) ( In contrast to rodent oocytes, in which protein synthesis is not required for entry into M phase (the pig oocyte requires protein synthesis to undergo meioticmaturation (The possible effect of estradiol on the synthesis of proteins that isvital for the cell cycle progression was evaluated in the present study by maturating oocytesin estradiol-free medium. When oocytes were cultured in presence of various concentrationsof an antiestrogen, no effect was observed in the percentage of oocytes that remained ingerminal vesicle stage or that reached metaphase II stage. Similar results were obtainedwhen an aromatase inhibitor was added to the maturation medium, which suggests thatestradiol is not involved in the protein synthesis required for the MPF activity. This findingis supported by studies with ovine oocytes (Such studies showedthat the nuclei of ovine oocytes could be removed at various times before GVBD withoutpreventing the changes in protein synthesis, concluding that the presence of the nucleus isnot 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 cytoplasmicmaturation by acting at the oocyte surface (in a non-genomicsignaling manner. In human oocytes, estradiol induced a rapid increase in free intracellularCa2+ concentration followed by a series of Ca2+ oscillations enhancing the cytoplasmicmaturation (However, the same effect was not observed in thepresent 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 itsactivation, have not been established. Nevertheless, the results of some studies suggest the M.A.N. Dode, C.N. Graves / Animal Reproduction Science 78 (2003) 99–110 requirement of steroid hormones for the MPN formation (the present study, the absence of estradiol had no significanteffect either on nuclear and cytoplasmic maturation or on the other characteristics studied.
The similar results obtained in the tamoxifen and aromatase inhibitor studies suggest thatestradiol 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 additionof estradiol. Whether the amounts used were effective in blocking estradiol action is notknown, however, it was expected, by studies with other cell types to be sufficient to competewith estradiol and bind with its receptors. Greater concentrations could not be used becausewhen 10−4 M of tamoxifen was tested in a preliminary study (data not shown) a detrimentaleffect on oocyte maturation occurred because all the oocytes were held at the germinalvesicle 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 toinhibit all the estradiol production. No measurement was made of the estradiol concentra-tion 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, personalcommunication).
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. Furtherstudies are needed to verify the involvement of estradiol in the ability of the oocytes to beactivated.
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 theestradiol in the follicular fluid is only needed to keep the oocyte in meiotic arrest and itis not required for maturation itself. It is important to consider also that these results donot eliminate the effect of estradiol in cleavage and embryonic development as well as theinvolvement of other steroid hormones in maturation. Clarification of these aspects willrequire further studies.
This project was partially funded by the University of Illinois Agriculture Experiment Station, Grant #ILLU-35-334. The authors thank the USDA Animal Program for the FSHand LH, Dr. Brodie, University of Maryland (Baltimore), for the aromatase inhibitor andExel Corp., Beardstown, IL, for furnishing the ovaries used in this study.
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