Specificity of Inhibition by Steroids of Porcine Oocyte Maturation In Vitro JOEL D. RICHTER AND ROBERT W. MCGAUGHEY Department of Zoology, Arizona State University, Tempe, Arizona 85281
ABSTRACT In order to examine the influence of several steroids on the process of oocyte maturation, denuded (adherent cumulus granulosa cells mechanically removed) and intact (cumulus granulosa cells left attached) porcine oocytes were cultured in the presence or absence of estradiol-l7P, estradiol-l7a, testosterone, cortisol, progesterone, or the nonsteroidal estrogen diethyl stilbestrol (all a t 10 pg/ml) in defined medium that contained either BSA or dextran. Estradiol-17P was the only steroid to exert a significant inhibitory effect on the maturation of denuded oocytes, and did so only in BSA supplemented medium. The inhibition was reversible in t h a t oocytes, cultured in steroid-free medium after initial culture in estradiol-17P medium, resumed meiotic maturation. Oocytes took up 3H-estradiol-17Pin both media, although less radiolabel entered oocytes in BSA supplemented medium. The majority of label in the oocytes, when cultured with either medium, was not displaced by excess radioinert estradiol-17P or progesterone, nor were the oocytes saturated even when cultured in M estradiol-17P. Autoradiography of sectioned oocytes after culture in 3H-estradiol-17Phas shown that there was no selective accumulation of silver grains over the germinal vesicle as was the case with granulosa cell nuclei. This observation suggests that estradiol-17P may not act a t the level of the oocyte nucleus. Meiotic maturation of oocytes has been shown to be regulated by a diversity of chemicals in various animals. In starfish, l-methyladenine stimulates germinal vesicle breakdown and chromosomal condensation (Kanatani et al., '69; Kanatani, '72); whereas amphibian oocyte maturation may be induced by progestins (Smith and Ecker, '71; Reynhout and Smith, '73; Smith, '751, calcium actiHvators (Wasserman and Masui, '751, or propanolol-like drugs (Baulieu et al., '78). Mammalian oocytes m a t u r e spontaneously i n chemically defined culture medium in the absence of apparent inducers (McGaughey, '77a,b). It therefore seems likely t h a t precocious maturation is specifically inhibited by a substance in the mammalian oocyte or follicle. It has been reported that a cyclic nucleotide (Wassarman and Turner, '76; Schultz and Wassarman, '771, a follicular fluid peptide (Tsafriri e t al., '76; Stone et al., '77), and an estrogen (McGaughey, '77a) are able to inhibit the spontaneous maturation of mammalian oocytes in vitro. J. EXP. ZOOL. (1979)209: 81-90.
Porcine oocytes have been shown to mature both in complex (McGaughey and Polge, '71; Tsafriri and Channing, '75) and minimal (McGaughey, '77a,b, '78) culture media, and minimal medium supplemented either with bovine serum albumin (BSA) (McGaughey, '77a) or dextran (McGaughey, '77b; McGaughey and Van Blerkom, '77) supports maturation of porcine oocytes. Steroid inhibition, however, has been previously demonstrated only in medium supplemented with BSA (McGaughey, '77a). This report examines the ability of estradiol-17p to inhibit meiotic maturation of pig oocytes in different culture media. Evidence presented here suggests that BSA is required for estradiol inhibition, and that other steroids fail t o elicit inhibition of denuded oocytes. We further show t h a t the uptake of tritium-labeled estradiol (at 3.3 x lo-' M) plateaus in porcine oocytes in two hours, that the oocytes are not saturated with high amounts of estradiol-l7P, and that the steroid does not selectively accumulate within germinal vesicles.
81
82
JOEL D. RICHTER AND ROBERT W. MCGAUGHEY MATERIALS AND METHODS
Collection of oocytes Porcine oocytes were collected a t slaughter and transported immediately to the laboratory. Oocytes from small antral follicles (1-2 mm in diameter) were liberated into culture medium by gently tearing the follicular wall. Only those oocytes t h a t had a uniform cumulus mass were selected for subsequent experiments. Some oocytes were mechanically denuded with a small bore pipette (McGaughey and Van Blerkom, '77) and washed twice in medium before culture. The transport of reproductive tracts, and the harvesting and selection of oocytes have been described previously (McGaughey, '77a,b, '78). Culture media and steroids
Two minimal culture media were employed that contained the same salts, but which differed in macromolecular supplement. BMOC-3 (Brinster, '71) contained BSA (4.5 mg/ml, Sigma), while a modification of this medium (McGaughey, '77b) contained dextran T-70 (4.5 mg/ml, Pharmacia). Experiments were performed with each medium in an attempt to determine the specificity of steroid-oocyte interactions with regard to culture conditions. Stock solutions of estradiol-l7P, estradiol1 7 q progesterone (all from Sigma) testosterone, or cortisol (both from Schwarz-Mann) were prepared by dissolving each steroid in 95% ethanol a t a concentration of 5 mg/ml. Diethyl stilbestrol (DES, from Sigma) was dissolved in benzene and ethanol (9:l) at a concentration of 5 mg/ml. Twenty microliters of each stock solution were pipetted into a sterile flask, the solvent allowed to evaporate, and 10 ml of the appropriate culture medium were added to give a final concentration of 10 pg/ ml. These solutions were stirred overnight a t 4°C.
riod of 31 to 33 hours. After culture, all oocytes were fixed with acetic acid-ethanol, air dried, stained with 0.5%basic fuchsin, and scored for stage of meiosis (McGaughey and Polge, '71). At least four replicates were performed for each treatment and control group. Data are expressed as percentages of maturation for each group, and statistical differences were assessed by the Duncan Multiple Range Tests (McGaughey, '77a), or by Student's t-test. Reversible inhibition by estradiol-17P In a series of separate experiments, oocytes were collected in control BMOC-3 (BSA) and transferred either to BMOC-3 containing estradiol-17P (10 pg/ml) or to control BMOC-3. After 24 hours of culture, representative oocytes from these two groups were air-dried and stained, and the remaining oocytes were transferred to fresh control BMOC-3 and cultured for an additional 24 hours, after which they were air-dried and stained. Uptake of 3H-estradiol by oocytes
Oocytes were collected in each of the two control media described above. For each experiment, ten denuded oocytes were cultured in 0.5 ml of the appropriate medium containing (2, 3, 6, 7, 16, 17) 3H-estradiol-17P(New England Nuclear, specific activity 152 Ci/ mmole) a t a concentration of 3.3 x 1 0 -M. ~ After various periods of culture oocytes were removed from t h e radioactive medium, washed three times in control medium, and transferred in 10 p l of medium to scintillation vials. Background radioactivity was determined with an aliquot (10 p1) of the third wash. Radioactivity was measured in a Beckman LSC-230 liquid scintillation counter a t a tritium efficiency of 45%.The displacement of 3H-estradiol from denuded oocytes was measured after it was determined from the experiments above that the oocytes reached an Culture conditions uptake plateau with 3H-estradiol after two Oocytes were harvested in the appropriate hours of culture. Oocytes were cultured for control medium and immediately transferred five hours in medium containing 3H-estradiol to the same medium containing one of the a t the same concentration a s above; then they above hormones. In a typical replicate experi- were removed, washed three times in control ment, ten oocytes were cultured in 0.5 ml of medium, and cultured for various additional medium in plastic culture dishes (Falcon times in medium containing either 3.6 x M 1008, Oxnard, California). The dishes were M radioinert estradiol-17P or 3.1 X placed in humidified desiccators which were radioinert progesterone. The degree to which oocytes are saturated gassed with an environment of 5% COz, 5%0 2 , and 90% N,. After sealing, the desiccators with estradiol was determined with labeled were placed in an incubator a t 37°C for a pe- and unlabeled estradiol-17P. Denuded oocytes
83
STEROID INHIBITION OF PORCINE OOCYTE MATURATION
were cultured for four hours in BMOC-3 (BSA) which contained 3.3 x M 3H-estradioland one of the following concentrations of radioM, 3.3 X M, inert estradiol: 0, 3.3 X 3.3 X M, or 3.3 X M. After culture, the oocytes were washed and radioactivity determined as described above. Autoradiography
according to Luft ('61). A Porter-Blum MT-1 ultramicrotome was used to prepare 0.5-pm sections which were mounted on glass slides, coated with Nuclear Track Emulsion (Kodak) and exposed for two weeks. The sections, stained with toluidine blue and methylene blue, were examined by light microscopy.
Intact and denuded oocytes were cultured in BMOC-3 that contained 1.6 x M 3Hestradiol (2.5 pCi/0.5 ml) for five hours. The oocytes were fixed according to Karnovsky ('651, post-fixed with osmium (l%, w/v), dehydrated in ethanol, and embedded in Epon
Steroid On Oocyte maturation The results of experiments in which intact oocytes were cultured in BMOC-3 or in modified BMOC-3 in the presence or absence of steroids or DES, are shown in table 1. Oocytes cultured in BMOC-3 (BSA) contain-
RESULTS
TABLE 1
Percentages of intact oocytes which matured during 31 to 33 hours of culture Mean percentage (2SE)of oocytes which matured '
Medium Treatment (10 pg/ml)
No. oocytes
Dia-M2
A1-M2
3520. 4 (a) 1220. 2 (a,b) 1621. 4 (a,b) 2121.5 (a,b) 2520. 5 (a,b) 1620. 1 (a,b) 1020.2 cb) 3 1 2 1.2 (x) 2220.6 (x) 1123. 2 (XI 1821.1 (XI 1 3 2 1.3 (XI
BMOC-3 (BSA)
None Progesterone Cortisol Testosterone Diethyl stilbestrol Estradiol- 170 Estradiol-170
135 36 58 37 32 37 111
6820.4 (A,B) 7121.1 (A,B) 3724.4 (B,C) 5421.2 (A,B,C) 8 2 2 0 . 1 (A) 5620.2 (A,B,C) 3220.9 (C)
Modified BMOC-3 (dextran)
None Progesterone Cortisol Testosterone Estradiol-17P
117 55 71 33 62
6320.3 53k0.2 3424.1 45kO.O 4520.0
(XI (X,Y) (Y)2 (X,Y) (X,Y)
' Dia-M2 refers to the stages of diakinesis to metaphase 2 and A1-M2 refers to the stages of anaphase 1 to metaphase 2. Mean percentages and SE were calculated from transformed angular data following statistical testing (cf. McGaughey, '77). Letters which differ for groups represent statistically different subsets in the statiatical test (p < 0.05). See text for a statistical explanation regarding the high SE for the cortisol group. TABLE 2
Percentages of denuded oocytes which matured during 31 to 33 hours of culture Mean percentages ( 5 SE)of mcytes which matured
Medium Treatment (10 pglml)
BMOC-3 (BSA)
None Progesterone Cortisol Testosterone Diethyl stilbestrol Estradiol- 17a Estradiol-17P
Modified BMOC-3 (dextran)
None Progesterone Cortisol Testosterone Estradiol- 17p
'See table 1 for further explanation
NO.oocytes
Dia-M2
A1-M2
147 62 59 64 31 32 111
4920.3 (A) 3820.6 (A) 4721.7 (A) 35k0.9 (A) 4520.2 (A) 5 0 k 1.3 (A) 14k0.3 (Bf
2020.1 (a) 920. 6 (a,b,c) 1 3 2 1.0 (a,b,c) 1 6 2 1.3 (a,b) 1020. 8 (a,b,c) 620. 7 (b,c) 320. 1 (c)
5020.7 2621.4 3420.0 5421.0 3922.5
2021. 1 (x) 520. 6 (XI 220. 2 (XI 6 2 0 . 3 (x) 3 5 0 . 1 (K)
85 48 64 48 43
(XI (XI
(XI (XI (X)
a4
JOEL D. RICHTER AND ROBERT W. MCGAUGHEY TABLE 3 Reversible nature of estradiol-17p inhibition of porcine oocytes Total hours of culture
Culture medium
24
BMOC-3 BMOC-3 BMOC-3 + estradiol I BMOC-3 + estradiol (24 hours) then BMOC-3 (24 hours)
Mean percentages (t SE) of oocytes which matured (Dia-MP)
No. examined
44t3 52k2 21t4 52k3
34 52 27
48
24 48
56
’ Estradiol refers to estradiol-17P at a concentration of 10 &g/ml. ’Significantly higher (p < 0.05) than BMOC-3 (24 hours), by Student’s t test. JSignificantly lower (p < 0.002)than the other three graupe, by Student’s t test.
W 0
t> 0 0
0
‘ 50 O01 04
0
1
2
3
4
5
6
7
8
HOURS
Fig. 1 Uptake of 3H-estradiol by denuded oocytes in BMOC-3 (top) and modified BMOC-3 (bottom). 00cytes were cultured in 3.3 x M Westradio1 ( 0 - 0 ) for t h e times shown. Once a n uptake plateau was M determined, oocytes were cultured for five hours in label as before, then transferred to either 3.7 X M radioinert progesterone (0-13,and cultured for the additional radioinert estradiol (0-0) or 3.1 x times shown.
ing estradiol-l7a, DES, testosterone, or cortisol matured at similar incidences statistically as compared with oocytes cultured in control medium. When intact oocytes were cultured in BMOC-3 (BSA), estradiol-17P inhibited maturation significantly (p < 0.05) as compared with DES, progesterone or controls.
The incidence of the first meiotic division was also significantly reduced by estradiol-17P. In modified BMOC-3, intact oocytes matured a t a reduced incidence in the presence of cortisol as compared with controls. However, due to the high degree of within-group variability among cortisol replicates (SE, 4.1%),
85
STEROID INHIBITION OF PORCINE OOCYTE MATURATION
250-
200-
~
Y
+ U
0 0
150-
0
c
\
I
n 0
100-
0 ’
I
0
3.3 10-9
3.3:
3.3: 10- 7
3.3:10-’
C O N C E N T R A T I O N (MI Of R A D I O I N E R T ESTRADIOL
Fig. 2 Saturation kinetics of oocytes with estradiol-17P. Oocytea were cultured for four hours in 3.3 x 10-9 M M radioinert estradiol-17P. estradiol-17P and 0, 3.3 X 10‘9M, 3.3 X 1 O P M, 3.3 X IO”M, or 3.3 X
and the accompanying heterogeneity of variances determined statistically for these groups, this statistical statement of significant difference for the cortisol group may not be valid. When the cortisol group was excluded from the Duncan Multiple Ranges Test, no significant differences were found among t h e remaining groups. No steroid significantly inhibited the first meiotic division of intact oocytes cultured in modified BMOC-3. The results of experiments with denuded oocytes cultured in the two media with and without steroids or DES and are shown in table 2. Denuded oocytes cultured in BMOC-3 (BSA) containing estradiol-17P matured a t a much lower incidence (p < 0.05) than did oocytes cultured in the same medium without steroid, with one of the other four steroids or with DES. The first meiotic division was inhibited by estradiol-17P and estradiol-17a as compared with controls (p < 0.05). In contrast, denuded oocytes matured equally well i n modified BMOC-3 (dextran) containing no steroid or any of the four steroids. The reversible nature of estradiol inhibition in BMOC-3 reported earlier (McGaughey, ‘77a) is confirmed here. It is seen in table 3 that estradiol inhibited oocyte maturation by 39% relative to controls during the 24-hour cultures. Oocytes, which were cultured in me-
3H-
dium containing estradiol for 24 hours and subsequently transferred to control medium and cultured for an additional 24 hours, matured a t the same incidence as oocytes which were cultured for 48 hours in steroidfree medium. Association of estradiol with oocytes The time course of the association of 3Hestradiol with denuded oocytes cultured in BMOC-3 and in modified BMOC-3 is shown in M 3Hfigure 1. When cultured in 3.3 x estradiol, uptake of the steroid reached a plateau by two hours. About 57% more label entered oocytes when cultured in modified BMOC-3 (275 dpm/lO oocytes) as compared with BMOC-3 (175 dpm/l0 oocytes). In separate experiments, oocytes were cultured for five hours in medium containing 3H-estradiol MI. These oocytes were then trans(3.3 X ferred to medium containing a 100 fold excess of radioinert estradiol or progesterone. Both steroids were able to displace some of the radiolabel in both culture media. The extent to which oocytes exhibit saturation kinetics with estradiol-17P is shown in figure 2. Oocytes, when cultured in medium containing a constant amount of labeled M) and increasing estradiol (3.3 X amounts of unlabeled estradiol (0, 3.3 x 10-9
86
JOEL D. RICHTER AND ROBERT W. McGAUGHEY
Fig. 3 Autoradiographs of granulosa cells (top) and a denuded oocyte (bottom). Cells were cultured for five hours in 2.5 FCi 3H-estradiol and processed for radiography a s described in MATERIALS AND METHODS. Oocyte germinal vesicle (GV), zona pellucida (ZP),and granulosa cell nuclei (N) are noted. In the oocyte, there is a silver grain ratio of 3:5 over t he germinal vesicle and cytoplasm, respectively. X 535.5.
STEROID INHIBITION OF PORCINE OOCYTE MATURATION
3.3 x 1 0 - 8 M, 3.3 x 10-7 M, 3.3 x 1 0 - 6 M) took up the same amount of labeled steroid regardless of the concentration of unlabeled steroid present. Therefore, after four hours of culture, oocytes were not saturated with estradiol even at the very high concentration Of 3.3 X 10-6M. The distribution of 3H-estradiol within oocytes was examined by autoradiography and is shown in figure 3. Silver grains appear to be randomly distributed over the cytoplasm, and there is no apparent selective concentration either in the germinal vesicle or a t the plasma membrane. When grain counts were made, there were fewer over the germinal vesicle than over any other part of the oocyte of comparable area (a ratio of about 3:5), excluding the zona pellucida. Radiographs were also prepared with granulosa cells, in which there is known to be an estradiol receptor (Richards, '751, for comparison to the radiographs of oocytes. It is readily apparent that many silver grains were concentrated over the nuclei when these cells were cultured under conditions identical to those employed with denuded oocytes. M,
DISCUSSION
Previous work has demonstrated that estradiol-17P reversibly inhibits the maturation of denuded porcine oocytes, and that progesterone will overcome this inhibitory effect (McGaughey, '77a). We have expanded this observation in the present study by determining that estradio:-17P, as compared with estradiol-17&,DES, progesterone, cortisol, and testosterone, is the most effective and consistent inhibitor of maturation for the porcine oocyte. In addition we have now demonstrated the following in respect to the inhibitory influence of estradiol-17P on porcine oocytes in vitro. (1)Inhibition by estradiol appears to be dependent upon t h e presence of a proteinaceous macromolecule (i.e., BSA in this study); particularly in the case of denuded oocytes. ( 2 ) Estradiol-17& at a sufficiently high concentration (i.e., 10 pg/ml), inhibits the maturation of intact porcine oocytes as well as denuded oocytes. (3) The nonsteroidal estrogen, DES, fails to inhibit maturation. (4) There is some stereospecificity involved since estradiol-17a does not inhibit overall maturation, but does inhibit the first meiotic division in denuded oocytes. A reasonable explanation for the different results obtained with media containing BSA
87
or dextran concerns the relative affinity of estradiol for these two macromolecules. We have observed that when BMOC-3 containing BSA and modified BMOC-3 containing dextran were incubated with 3H-estradiol (3.3 x M) and subsequently dialyzed against distilled water for 36 hours a t 4"C, 66% of the total estradiol was bound by the BSA of BMOC-3 while no steroid was bound by dextran (results not shown). This observation suggests that BSA might act in one of two ways to facilitate the inhibitory influence of estradiol on porcine oocytes. I t may increase the solubility of estradiol by binding it, which could result in a higher effective concentration of the steroid in BMOC-3 than in modified BMOC-3. BSA may also act as a carrier molecule to transport estradiol into the oocyte since i t is known that proteins including BSA can enter mammalian oocytes (Glass, '63, '71). In light of our association-dissociation studies (see below), the present results with denuded oocytes (cf. table 2 ) are more consistent with the latter explanation. At present, no clear explanation can be offered for the observation that cortisol may have inhibited the maturation (i.e., diakinesis to metaphase 2) of intact oocytes in modified BMOC-3 (cf. statistical caveat in RESULTS section). However, denuded oocytes were not inhibited by cortisol. The possibility therefore exists that cortisol may have interacted with the surrounding granulosa cells, in the case of intact oocytes, and brought about the observed inhibition indirectly. McNatty e t al. ('75) found t h a t fluid from small follicles contained as much as approximately 300 ng of estradiol per ml; while nearly 33 times this concentration (i.e., 10 p g per ml) was employed in our experiments to inhibit the maturation in vitro of porcine oocytes. In an earlier study (McGaughey, '77a), however, 1.0 p g of estradiol per ml also caused a statistically significant inhibition of maturation in denuded porcine oocytes in vitro. Currently it is not known what proportion of follicular fluid steroids are bound to macromolecules (Cook et al., '77; Mahajan and Little, '78), or what macromolecules might be required for the transport of steroids from one follicular compartment to another (Baird, '77; Moor, '77). In our present experiments in which estradiol (10.0 pg/ml) inhibited maturation in BMOC-3, the BSA concentration was only 4.5 mg per ml as compared with nearly ten times that concentration of albu-
88
JOEL D. RICHTER AND ROBERT W. MCGAUGHEY
min known to be present in porcine follicular fluids (McGaughey, '75). The possibility exists t h a t certain macromolecules of follicular fluid, particularly the nonspecific steroidbinding molecule albumin, might facilitate the interactions between steroids and cells, including the oocyte, of the follicle. Thus, although the concentration of estradiol presently employed to examine the inhibition of maturation in denuded porcine oocytes might be considered high as compared with steroid concentrations present in follicular fluid, i t should be borne in mind t h a t the minimal media, BMOC-3 with BSA or dextran, probably exhibit very little capacity for steroid binding and transport in comparison to antral fluids. In our experiments we observed that the uptake of 3H-estradiol by porcine oocytes reaches a plateau within two hours of culture when the steroid concentration is well below that found in the fluid of porcine follicles. The concentration of 3H-estradiol employed in M) was apthese experiments Le., 3.3 x proximately two orders of magnitude lower than has been reported for antral fluid of porcine follicles (Hunter et al., '76). Our data relating to the dissociation of 3H-estradiol show that the dissociation of this steroid is much slower in the presence of either radioinert estradiol or progesterone than is its initial association with denuded oocytes. The association of estradiol with denuded oocytes is quantitatively greater Le., 57% more estradiol uptake) in medium containing dextran as compared with medium containing BSA (cf. fig. 1).This observation may be related to the fact mentioned above that BSA binds estradiol significantly relative to dextran. It would appear that a certain amount of BSAbound estradiol may not be available for association with denuded oocytes. Even after a 3-hour culture in excess radioinert steroid, the vast majority of initially associated 3Hestradiol remains associated with the oocytes. This indicates that the oocytes have a very high capacity for steroids which is confirmed by data presented in figure 2. The same amount of labeled estradiol enters oocytes over a 1,000-fold range of excess unlabeled estradiol. The presence of large numbers of lipid vesicles (Szollosi, '72) would be expected to partition large amounts of steroids nonspecifically, which greatly complicates the search for estradiol receptors, if in fact they are present in oocytes.
We observed by autoradiography that in contrast to porcine cumulus granulosa cells which selectively accumulated radiolabeled estradiol within their nuclei, porcine oocytes did not exhibit selectively localized silver grains over their germinal vesicles. Cytological methods required in the autoradiographic experiments of the present study, particularly the dehydration steps needed for preparation of plastic-embedded oocytes, would be expected to result in the extraction of some of the associated 3H-estradiol. Therefore, the amounts of radiolabel observed in these studies would be expected to represent an underestimate of the total steroid associated with the oocytes. Whether it was unaltered estradiol-17P or some metabolite of this steroid which was associated with and inhibited the maturation of porcine oocytes in this study is not yet clear. In a different system, the induction of maturation in the amphibian oocyte by progesterone, it is known that these oocytes can metabolize steroids and that the metabolites of progesterone maintain their inductive activi t y (Reynhout and Smith, '73). The ability of porcine oocytes to metabolize steroids is not currently known. Our present results, therefore, cannot be interpreted as evidence for the presence of specific receptors for estradiol-17P in porcine oocytes. The nonsteroidal estrogen diethyl stilbestrol, which binds estradiol receptors in other systems (Ruh and Baudendistel, '771, failed to elicit an inhibitory effect on maturation. We have also cultured denuded oocytes in medium containing both estradiol-17P and the estrogen antagonist nafoxidine (Lazier and Alford, '77) in a n attempt to preclude inhibition. However, nafoxidine, a t equal or greater concentrations as estradiol, caused cytoplasmic shrinkage and aberrant chromosomal configurations. Furthermore, the large steroid capacity of the oocyte makes the search for low capacity, high affinity receptors extremely difficult. Our autoradiographic observations could be interpreted to suggest the possibility that estradiol might act on porcine oocytes at a cellular site other than the nucleus. In another system, it is known that steroids can alter the ion permeability of amphibian oocyte membranes (Morrill et al., '77; O'Connor et al., '77) and that this alteration leads to maturation (Baulieu et al., '78). It is also known that steroids can influence the expression of cyclic
STEROID INHIBITION OF PORCINE OOCYTE MATURATION
AMP (Greengard, '78). The ability of dibutyryl cyclic AMP to inhibit the maturation of mouse oocytes is well documented (Wassarman and Turner, '76; Stern and Wassarman, '74); and the possibility that estradiol may act on porcine oocytes indirectly by means of the intermediate, cyclic AMP, is currently being examined in our laboratory. Taken together, we feel that although the possibility of estradiol receptors does exist, our data indicate that the specific action of the steroid may be a t an extranuclear site, perhaps the membrane. Recently it was reported that estradiol-17P does not inhibit the maturation of mouse oocytes in vitro (Eppig and Koide, '78). We have also observed that estradiol- 17p, even at the very high concentration of 25 pg/ml, does not decrease the incidence of maturation in mouse oocytes (unpublished observations). At present, the difference in response to estradiol between mouse and porcine oocytes may be ascribed to several explanations. Mouse oocytes used for studies of maturation in vitro are collected from large, preovulatory follicles (Donahue, '68; Schultz and Wassarman, '771, whereas porcine oocytes routinely have been harvested from small ovarian follicles (McGaughey, '78). Mouse oocytes selected for culture are easily denuded of their loosely associated cumulus granulosa cells (Donahue, '681, whereas porcine oocytes from small follicles exhibit very tightly adherent cumulus granulosa and corona radiata cells which are anchored to the oocytes by means of cytoplasmic projections (McGaughey, '78). In addition, the mouse oocyte exhibits a very short germinal vesicle stage in culture (i.e., approximately 2 hours; Donahue, '68), while the germinal vesicle of the porcine oocyte remains intact for approximately 10 to 15 hours prior to the resumption of meiosis in vitro (McGaughey and Polge, '71; McGaughey, '78). It therefore appears probable that mouse oocytes and porcine oocytes might constitute rather different physiological models regarding follicular stage and the influence of steroids on maturation in vitro. Whether the maturation of mammalian oocytes in vitro (i.e., spontaneous maturation) represents a normal physiological process or a degenerative artificial phenomenon has been questioned (e.g., Eppig and Koide, '78). Although some apparently normal fetuses have been obtained from mouse oocytes which were matured in vitro (Cross and Brinster, '70),
89
other studies have reported t h a t oocytes matured in vitro do not have normal capacity for further development (Niwa and Chang, '75; Moor and Trounson, '77). In contrast we recently demonstrated t h a t rabbit oocytes are capable of fertilization and preimplantation development in vivo following maturation in chemically defined medium in vitro, and that the patterns of polypeptide synthesis were comparable for rabbit oocytes matured in vivo and in vitro (Van Blerkom and McGaughey, '78a,b). Additional work is necessary to further assess the level of normalcy exhibited by mammalian oocytes matured in vitro, and to characterize the physiological requirements for maturation in vivo. The application of culture procedures currently appears to be necessary for the experimental dissection of the ovarian follicular components which very probably influence the maturation of mammalian oocytes. ACKNOWLEDGMENTS
The authors are grateful to Cudahy Foods of Phoenix, Arizona, for supplying the porcine reproductive tracts and to t h e Upjohn Company for supplying the nafoxidine. This study was supported in part by Grant HD06532 from the NIH and by the Arizona State University Research Enrichment Program. LITERATURE CITED Baird, D. T. 1977 Evidence in uiuo for the two-cell hypothesis of oestrogen synthesis by the sheep Graafian follicle. J. Reprod. Fert., 50: 183-185. Baulieu, E. E., F. Godeau, M. Schorderet and S. SchoderetSlatkin 1978 Steroid-induced meiotic division in Xenopus laeuis, oocytes: Surface and calcium. Nature, 275: 593. Brinster, R. L. 1971 Measuring embryonic enzyme activity. In: Methods in Mammalian Embryology. J. C. Daniel, Jr., ed. W. H. Freeman and Co., San Francisco, pp. 215-227. Cook, B., R. H. F. Hunter and A. S. Kelley 1977 Steroidbinding proteins in follicular fluid and peripheral plasma from pigs, cows and sheep. J. Reprod. Fert., 50: 65-71. Cross, P. C., and R. L. Brinster 1970 In uitro development of mouse oocytes. Biol. Reprod., 3: 298-307. Donahue, R. P. 1968 Maturation of the mouse oocyte in uitro. I. Sequence and timing of nuclear progression. J. Exp. Zool., 169: 237-250. Eppig, J. J., and S. L. Koide 1978 Effects of progesterone and oestradiol-17P on the spontaneous meiotic maturation of mouse oocytes. J. Reprod. Fert., 53: 99-101. Glass, L. 1963 Transfer of native and foreign serum antigens to oviducal mouse eggs. Am. Zool., 3: 135-156. 1971 Transmission of maternal protein into oocytes. In: Advances in the Biosciences 6. Schering Symposium on Intrinsic and Extrinsic Factors in Early Mammalian Development. G. Rasp& ed. Pergamon Press, New York, pp. 29-67.
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JOEL D. RICHTER AND ROBERT W. MCGAUGHEY
Greengard, P. 1978 Phosphorylated proteins as physiological effectors. Science, 199: 146-152. Hunter, R. H. F., B. Cook and T. G. Baker 1976 Dissociation of response to injected gonadotropin between the Graafian follicle and oocyte in pigs. Nature, 260: 156-158. Kanatani, H. 1972 Adenine derivatives and oocyte maturation in starfishes. In: Oogenesis. J. D. Biggers and A. W. Schuetz, eds. University Park Press, Baltimore, pp. 459-478. Kanatani, H., H. Shirai, K. Nakanisha and T. Kurokawa 1969 Isolation andidentification of meiosis inducingsubstance in starfish Asterias arnurensis. Nature, 221: 273-274. Karnovsky, M. J. 1965 A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell. Biol., 27: 137A. Lazier, C. B., and W. S. Alford 1977 Interaction of the antioestrogen, Nafoxidine hydrochloride, with the soluble nuclear oestradiol binding protein in chick liver. Biochem. J., 164: 659. Luft, J. H. 1961 Improvement in epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 9: 409-414. Mahajan, D., and A. Little 1978 Specific cortisol binding protein in porcine follicular fluid. Biol. Reprod., 18: 834-842. McGaughey, R. W. 1975 A comparison of the fluids from small and large ovarian follicles of the pig. Biol. Reprod., 13: 147-153. 1977a The culture of pigoocytes in minimal medium, and the influence of progesterone and estradiol-17P on meiotic maturation. Endocr., ZOO: 39-45. 197713 The maturation of porcine oocytes in minimal, defined culture media with varied macromolecular supplements and varied osmolarity. Exp. Cell Res., 109: 25-30. 1978 In uitro oocyte maturation. In: Methods in Mammalian Reproduction. J. C. Daniel, Jr., ed. Academic Press, New York, pp. 1-19. McGaughey, R. W., and C. Polge 1971 Cytogenetic analysis of pig oocytes maturedin uitro. J. Exp. Zool., 176: 383-395. McGaughey, R. W., and J. Van Blerkom 1977 Patterns of polypeptide synthesis of porcine oocytes during maturation. Devel. Biol., 56: 241-254. McNatty, K. P., W. M. Hunter, A. S. McNeilly and R. S. Sawers 1975 Changes in the concentration of pituitary and steroid hormones in the follicular fluid of human Graafian follicles throughout t he menstrual cycle. J. Endocr., 64: 555-571. Moor, R. M. 1977 Sites of steroid productions in ovine Graafian follicles in culture. J. Endocr., 73: 143-150. Moor, R. M., and A. 0. Trounson 1977 Hormonal and follicular factors affecting maturation of sheep oocytes in uitro and their subsequent developmental capacity. J. Reprod. Fert., 49: 101-109. Morrill, G. A., F. Schatz, A. Kostellow and J. Poupko 1977 Changes in cyclic AMP levels in the amphibian ovarian follicle following progesterone induction of meiotic maturation. Differentiation, 8: 97-104. Niwa, K., and C. K. Chang 1975 Fertilization of rat eggs in uitro a t various times before and after ovulation with
special reference to fertilization of ovarian oocytes matured in culture. J. Reprod. Fert., 43: 435-451. O'Connor, C. M., K. M. Robinson and L. D. Smith 1977 Calcium, potassium, and sodium exchange by full-grown and maturing Xenopus Zaeuis oocytes. Devel. Biol., 61: 28-40. Reynhout, J. K., and L. D. Smith 1973 Evidence for steroid metabolism during the in uitro induction of maturation in oocytes of Rana pipiens. Devel. Biol., 30: 392-402. Richards, J. S. 1975 Estradiol receptor content in rat granulosa cells during follicular development: Modification by estradiol and gonadotropins. Endocr., 99: 1174-1184. wruh, T. S., and L. J. Baudendistel 1977 Different nuclear binding sites for antiestrogen and estrogen receptor complexes. Endocr., ZOO: 420-426. Schultz, R. M., and P. M. Wassarman 1977 Biochemical studies of mammalian oogenesis: Protein synthesis during oocyte growth and meiotic maturation in the mouse. J. Cell Sci., 24: 169-194. Smith, L. D. 1975 Molecular events during oocyte maturation. In: The Biochemistry of Animal Development. R. Weher, ed. Academic Press, New York, Vol. 3, pp. 1-46. Smith, L. D., and R. Ecker 1971 The interaction of steroids with Rana pzpiens oocytes in the induction of maturation. Devel. Biol., 25: 237-247. Stern, S., and P. M. Wassarman 1974 Meiotic maturation of the mammalian oocyte in vitro. Effect of dibutyryl cyclic AMP on protein synthesis. J. Exp. Zool., 189: 275-281. Stone, S., S. Pomerantz, A. Kripner and C. Channing 1977 Purification of a porcine follicular fluid meiosis inhibitor and demonstration of reversibility of inhibitory action upon porcine oocyte maturation in culture. J. Cell Biol., 77: 174a. Szollosi, D. 1972 Changes of some cell organelles during oogenesis inmammals. In: Oogenesis. J. D. Biggers and A. W. Schuetz, eds. University Park Press, Baltimore, pp. 47-64. Tsafriri, A,, and C. Channing 1975 Influence of follicular maturation and culture conditions on meiosis of pig oocytes in uitro. J. Reprod. Fert., 43: 149-152. Tsafriri, A., S. Pomerantz and C. Channing 1976 Inhibition of oocyte maturation by porcine follicular fluid: Partial characterization of the inhibitor. Biol. Reprod., 4: 511-516. Van Blerkom, J., and R. W. McGaughey 1978a Molecular differentiation of the rabbit ovum. I. During oocyte maturation in uiuo and in uitro. Devel. Biol., 63: 139-150. 1978b Molecular differentiation of the rabbit ovum. 11. During preimplantation development of in uiuo and in uitro matured oocytes. Devel. Biol., 63: 151-164. Wassarman, P. M., and P. Turner 1976 Effect of dithiothreitol on meiotic maturation of mouse oocytes in vitro: Dependence of the effect of N6, O2-dibutyryl adenosine3',5' cyclic monophosphate. J. Exp. Zool., 196: 183-188. Wasserman, R., and Y. Masui 1975 Initiation of meiotic maturation of Xenopus Zaeuis oocytes by the combination of divalent cations and Inophore A23187. J. Exp. Zool., 193: 369-375.
ABSTRACT In order to examine the influence of several steroids on the process of oocyte maturation, denuded (adherent cumulus granulosa cells mechanically removed) and intact (cumulus granulosa cells left attached) porcine oocytes were cultured in the presence or absence of estradiol-l7P, estradiol-l7a, testosterone, cortisol, progesterone, or the nonsteroidal estrogen diethyl stilbestrol (all a t 10 pg/ml) in defined medium that contained either BSA or dextran. Estradiol-17P was the only steroid to exert a significant inhibitory effect on the maturation of denuded oocytes, and did so only in BSA supplemented medium. The inhibition was reversible in t h a t oocytes, cultured in steroid-free medium after initial culture in estradiol-17P medium, resumed meiotic maturation. Oocytes took up 3H-estradiol-17Pin both media, although less radiolabel entered oocytes in BSA supplemented medium. The majority of label in the oocytes, when cultured with either medium, was not displaced by excess radioinert estradiol-17P or progesterone, nor were the oocytes saturated even when cultured in M estradiol-17P. Autoradiography of sectioned oocytes after culture in 3H-estradiol-17Phas shown that there was no selective accumulation of silver grains over the germinal vesicle as was the case with granulosa cell nuclei. This observation suggests that estradiol-17P may not act a t the level of the oocyte nucleus. Meiotic maturation of oocytes has been shown to be regulated by a diversity of chemicals in various animals. In starfish, l-methyladenine stimulates germinal vesicle breakdown and chromosomal condensation (Kanatani et al., '69; Kanatani, '72); whereas amphibian oocyte maturation may be induced by progestins (Smith and Ecker, '71; Reynhout and Smith, '73; Smith, '751, calcium actiHvators (Wasserman and Masui, '751, or propanolol-like drugs (Baulieu et al., '78). Mammalian oocytes m a t u r e spontaneously i n chemically defined culture medium in the absence of apparent inducers (McGaughey, '77a,b). It therefore seems likely t h a t precocious maturation is specifically inhibited by a substance in the mammalian oocyte or follicle. It has been reported that a cyclic nucleotide (Wassarman and Turner, '76; Schultz and Wassarman, '771, a follicular fluid peptide (Tsafriri e t al., '76; Stone et al., '77), and an estrogen (McGaughey, '77a) are able to inhibit the spontaneous maturation of mammalian oocytes in vitro. J. EXP. ZOOL. (1979)209: 81-90.
Porcine oocytes have been shown to mature both in complex (McGaughey and Polge, '71; Tsafriri and Channing, '75) and minimal (McGaughey, '77a,b, '78) culture media, and minimal medium supplemented either with bovine serum albumin (BSA) (McGaughey, '77a) or dextran (McGaughey, '77b; McGaughey and Van Blerkom, '77) supports maturation of porcine oocytes. Steroid inhibition, however, has been previously demonstrated only in medium supplemented with BSA (McGaughey, '77a). This report examines the ability of estradiol-17p to inhibit meiotic maturation of pig oocytes in different culture media. Evidence presented here suggests that BSA is required for estradiol inhibition, and that other steroids fail t o elicit inhibition of denuded oocytes. We further show t h a t the uptake of tritium-labeled estradiol (at 3.3 x lo-' M) plateaus in porcine oocytes in two hours, that the oocytes are not saturated with high amounts of estradiol-l7P, and that the steroid does not selectively accumulate within germinal vesicles.
81
82
JOEL D. RICHTER AND ROBERT W. MCGAUGHEY MATERIALS AND METHODS
Collection of oocytes Porcine oocytes were collected a t slaughter and transported immediately to the laboratory. Oocytes from small antral follicles (1-2 mm in diameter) were liberated into culture medium by gently tearing the follicular wall. Only those oocytes t h a t had a uniform cumulus mass were selected for subsequent experiments. Some oocytes were mechanically denuded with a small bore pipette (McGaughey and Van Blerkom, '77) and washed twice in medium before culture. The transport of reproductive tracts, and the harvesting and selection of oocytes have been described previously (McGaughey, '77a,b, '78). Culture media and steroids
Two minimal culture media were employed that contained the same salts, but which differed in macromolecular supplement. BMOC-3 (Brinster, '71) contained BSA (4.5 mg/ml, Sigma), while a modification of this medium (McGaughey, '77b) contained dextran T-70 (4.5 mg/ml, Pharmacia). Experiments were performed with each medium in an attempt to determine the specificity of steroid-oocyte interactions with regard to culture conditions. Stock solutions of estradiol-l7P, estradiol1 7 q progesterone (all from Sigma) testosterone, or cortisol (both from Schwarz-Mann) were prepared by dissolving each steroid in 95% ethanol a t a concentration of 5 mg/ml. Diethyl stilbestrol (DES, from Sigma) was dissolved in benzene and ethanol (9:l) at a concentration of 5 mg/ml. Twenty microliters of each stock solution were pipetted into a sterile flask, the solvent allowed to evaporate, and 10 ml of the appropriate culture medium were added to give a final concentration of 10 pg/ ml. These solutions were stirred overnight a t 4°C.
riod of 31 to 33 hours. After culture, all oocytes were fixed with acetic acid-ethanol, air dried, stained with 0.5%basic fuchsin, and scored for stage of meiosis (McGaughey and Polge, '71). At least four replicates were performed for each treatment and control group. Data are expressed as percentages of maturation for each group, and statistical differences were assessed by the Duncan Multiple Range Tests (McGaughey, '77a), or by Student's t-test. Reversible inhibition by estradiol-17P In a series of separate experiments, oocytes were collected in control BMOC-3 (BSA) and transferred either to BMOC-3 containing estradiol-17P (10 pg/ml) or to control BMOC-3. After 24 hours of culture, representative oocytes from these two groups were air-dried and stained, and the remaining oocytes were transferred to fresh control BMOC-3 and cultured for an additional 24 hours, after which they were air-dried and stained. Uptake of 3H-estradiol by oocytes
Oocytes were collected in each of the two control media described above. For each experiment, ten denuded oocytes were cultured in 0.5 ml of the appropriate medium containing (2, 3, 6, 7, 16, 17) 3H-estradiol-17P(New England Nuclear, specific activity 152 Ci/ mmole) a t a concentration of 3.3 x 1 0 -M. ~ After various periods of culture oocytes were removed from t h e radioactive medium, washed three times in control medium, and transferred in 10 p l of medium to scintillation vials. Background radioactivity was determined with an aliquot (10 p1) of the third wash. Radioactivity was measured in a Beckman LSC-230 liquid scintillation counter a t a tritium efficiency of 45%.The displacement of 3H-estradiol from denuded oocytes was measured after it was determined from the experiments above that the oocytes reached an Culture conditions uptake plateau with 3H-estradiol after two Oocytes were harvested in the appropriate hours of culture. Oocytes were cultured for control medium and immediately transferred five hours in medium containing 3H-estradiol to the same medium containing one of the a t the same concentration a s above; then they above hormones. In a typical replicate experi- were removed, washed three times in control ment, ten oocytes were cultured in 0.5 ml of medium, and cultured for various additional medium in plastic culture dishes (Falcon times in medium containing either 3.6 x M 1008, Oxnard, California). The dishes were M radioinert estradiol-17P or 3.1 X placed in humidified desiccators which were radioinert progesterone. The degree to which oocytes are saturated gassed with an environment of 5% COz, 5%0 2 , and 90% N,. After sealing, the desiccators with estradiol was determined with labeled were placed in an incubator a t 37°C for a pe- and unlabeled estradiol-17P. Denuded oocytes
83
STEROID INHIBITION OF PORCINE OOCYTE MATURATION
were cultured for four hours in BMOC-3 (BSA) which contained 3.3 x M 3H-estradioland one of the following concentrations of radioM, 3.3 X M, inert estradiol: 0, 3.3 X 3.3 X M, or 3.3 X M. After culture, the oocytes were washed and radioactivity determined as described above. Autoradiography
according to Luft ('61). A Porter-Blum MT-1 ultramicrotome was used to prepare 0.5-pm sections which were mounted on glass slides, coated with Nuclear Track Emulsion (Kodak) and exposed for two weeks. The sections, stained with toluidine blue and methylene blue, were examined by light microscopy.
Intact and denuded oocytes were cultured in BMOC-3 that contained 1.6 x M 3Hestradiol (2.5 pCi/0.5 ml) for five hours. The oocytes were fixed according to Karnovsky ('651, post-fixed with osmium (l%, w/v), dehydrated in ethanol, and embedded in Epon
Steroid On Oocyte maturation The results of experiments in which intact oocytes were cultured in BMOC-3 or in modified BMOC-3 in the presence or absence of steroids or DES, are shown in table 1. Oocytes cultured in BMOC-3 (BSA) contain-
RESULTS
TABLE 1
Percentages of intact oocytes which matured during 31 to 33 hours of culture Mean percentage (2SE)of oocytes which matured '
Medium Treatment (10 pg/ml)
No. oocytes
Dia-M2
A1-M2
3520. 4 (a) 1220. 2 (a,b) 1621. 4 (a,b) 2121.5 (a,b) 2520. 5 (a,b) 1620. 1 (a,b) 1020.2 cb) 3 1 2 1.2 (x) 2220.6 (x) 1123. 2 (XI 1821.1 (XI 1 3 2 1.3 (XI
BMOC-3 (BSA)
None Progesterone Cortisol Testosterone Diethyl stilbestrol Estradiol- 170 Estradiol-170
135 36 58 37 32 37 111
6820.4 (A,B) 7121.1 (A,B) 3724.4 (B,C) 5421.2 (A,B,C) 8 2 2 0 . 1 (A) 5620.2 (A,B,C) 3220.9 (C)
Modified BMOC-3 (dextran)
None Progesterone Cortisol Testosterone Estradiol-17P
117 55 71 33 62
6320.3 53k0.2 3424.1 45kO.O 4520.0
(XI (X,Y) (Y)2 (X,Y) (X,Y)
' Dia-M2 refers to the stages of diakinesis to metaphase 2 and A1-M2 refers to the stages of anaphase 1 to metaphase 2. Mean percentages and SE were calculated from transformed angular data following statistical testing (cf. McGaughey, '77). Letters which differ for groups represent statistically different subsets in the statiatical test (p < 0.05). See text for a statistical explanation regarding the high SE for the cortisol group. TABLE 2
Percentages of denuded oocytes which matured during 31 to 33 hours of culture Mean percentages ( 5 SE)of mcytes which matured
Medium Treatment (10 pglml)
BMOC-3 (BSA)
None Progesterone Cortisol Testosterone Diethyl stilbestrol Estradiol- 17a Estradiol-17P
Modified BMOC-3 (dextran)
None Progesterone Cortisol Testosterone Estradiol- 17p
'See table 1 for further explanation
NO.oocytes
Dia-M2
A1-M2
147 62 59 64 31 32 111
4920.3 (A) 3820.6 (A) 4721.7 (A) 35k0.9 (A) 4520.2 (A) 5 0 k 1.3 (A) 14k0.3 (Bf
2020.1 (a) 920. 6 (a,b,c) 1 3 2 1.0 (a,b,c) 1 6 2 1.3 (a,b) 1020. 8 (a,b,c) 620. 7 (b,c) 320. 1 (c)
5020.7 2621.4 3420.0 5421.0 3922.5
2021. 1 (x) 520. 6 (XI 220. 2 (XI 6 2 0 . 3 (x) 3 5 0 . 1 (K)
85 48 64 48 43
(XI (XI
(XI (XI (X)
a4
JOEL D. RICHTER AND ROBERT W. MCGAUGHEY TABLE 3 Reversible nature of estradiol-17p inhibition of porcine oocytes Total hours of culture
Culture medium
24
BMOC-3 BMOC-3 BMOC-3 + estradiol I BMOC-3 + estradiol (24 hours) then BMOC-3 (24 hours)
Mean percentages (t SE) of oocytes which matured (Dia-MP)
No. examined
44t3 52k2 21t4 52k3
34 52 27
48
24 48
56
’ Estradiol refers to estradiol-17P at a concentration of 10 &g/ml. ’Significantly higher (p < 0.05) than BMOC-3 (24 hours), by Student’s t test. JSignificantly lower (p < 0.002)than the other three graupe, by Student’s t test.
W 0
t> 0 0
0
‘ 50 O01 04
0
1
2
3
4
5
6
7
8
HOURS
Fig. 1 Uptake of 3H-estradiol by denuded oocytes in BMOC-3 (top) and modified BMOC-3 (bottom). 00cytes were cultured in 3.3 x M Westradio1 ( 0 - 0 ) for t h e times shown. Once a n uptake plateau was M determined, oocytes were cultured for five hours in label as before, then transferred to either 3.7 X M radioinert progesterone (0-13,and cultured for the additional radioinert estradiol (0-0) or 3.1 x times shown.
ing estradiol-l7a, DES, testosterone, or cortisol matured at similar incidences statistically as compared with oocytes cultured in control medium. When intact oocytes were cultured in BMOC-3 (BSA), estradiol-17P inhibited maturation significantly (p < 0.05) as compared with DES, progesterone or controls.
The incidence of the first meiotic division was also significantly reduced by estradiol-17P. In modified BMOC-3, intact oocytes matured a t a reduced incidence in the presence of cortisol as compared with controls. However, due to the high degree of within-group variability among cortisol replicates (SE, 4.1%),
85
STEROID INHIBITION OF PORCINE OOCYTE MATURATION
250-
200-
~
Y
+ U
0 0
150-
0
c
\
I
n 0
100-
0 ’
I
0
3.3 10-9
3.3:
3.3: 10- 7
3.3:10-’
C O N C E N T R A T I O N (MI Of R A D I O I N E R T ESTRADIOL
Fig. 2 Saturation kinetics of oocytes with estradiol-17P. Oocytea were cultured for four hours in 3.3 x 10-9 M M radioinert estradiol-17P. estradiol-17P and 0, 3.3 X 10‘9M, 3.3 X 1 O P M, 3.3 X IO”M, or 3.3 X
and the accompanying heterogeneity of variances determined statistically for these groups, this statistical statement of significant difference for the cortisol group may not be valid. When the cortisol group was excluded from the Duncan Multiple Ranges Test, no significant differences were found among t h e remaining groups. No steroid significantly inhibited the first meiotic division of intact oocytes cultured in modified BMOC-3. The results of experiments with denuded oocytes cultured in the two media with and without steroids or DES and are shown in table 2. Denuded oocytes cultured in BMOC-3 (BSA) containing estradiol-17P matured a t a much lower incidence (p < 0.05) than did oocytes cultured in the same medium without steroid, with one of the other four steroids or with DES. The first meiotic division was inhibited by estradiol-17P and estradiol-17a as compared with controls (p < 0.05). In contrast, denuded oocytes matured equally well i n modified BMOC-3 (dextran) containing no steroid or any of the four steroids. The reversible nature of estradiol inhibition in BMOC-3 reported earlier (McGaughey, ‘77a) is confirmed here. It is seen in table 3 that estradiol inhibited oocyte maturation by 39% relative to controls during the 24-hour cultures. Oocytes, which were cultured in me-
3H-
dium containing estradiol for 24 hours and subsequently transferred to control medium and cultured for an additional 24 hours, matured a t the same incidence as oocytes which were cultured for 48 hours in steroidfree medium. Association of estradiol with oocytes The time course of the association of 3Hestradiol with denuded oocytes cultured in BMOC-3 and in modified BMOC-3 is shown in M 3Hfigure 1. When cultured in 3.3 x estradiol, uptake of the steroid reached a plateau by two hours. About 57% more label entered oocytes when cultured in modified BMOC-3 (275 dpm/lO oocytes) as compared with BMOC-3 (175 dpm/l0 oocytes). In separate experiments, oocytes were cultured for five hours in medium containing 3H-estradiol MI. These oocytes were then trans(3.3 X ferred to medium containing a 100 fold excess of radioinert estradiol or progesterone. Both steroids were able to displace some of the radiolabel in both culture media. The extent to which oocytes exhibit saturation kinetics with estradiol-17P is shown in figure 2. Oocytes, when cultured in medium containing a constant amount of labeled M) and increasing estradiol (3.3 X amounts of unlabeled estradiol (0, 3.3 x 10-9
86
JOEL D. RICHTER AND ROBERT W. McGAUGHEY
Fig. 3 Autoradiographs of granulosa cells (top) and a denuded oocyte (bottom). Cells were cultured for five hours in 2.5 FCi 3H-estradiol and processed for radiography a s described in MATERIALS AND METHODS. Oocyte germinal vesicle (GV), zona pellucida (ZP),and granulosa cell nuclei (N) are noted. In the oocyte, there is a silver grain ratio of 3:5 over t he germinal vesicle and cytoplasm, respectively. X 535.5.
STEROID INHIBITION OF PORCINE OOCYTE MATURATION
3.3 x 1 0 - 8 M, 3.3 x 10-7 M, 3.3 x 1 0 - 6 M) took up the same amount of labeled steroid regardless of the concentration of unlabeled steroid present. Therefore, after four hours of culture, oocytes were not saturated with estradiol even at the very high concentration Of 3.3 X 10-6M. The distribution of 3H-estradiol within oocytes was examined by autoradiography and is shown in figure 3. Silver grains appear to be randomly distributed over the cytoplasm, and there is no apparent selective concentration either in the germinal vesicle or a t the plasma membrane. When grain counts were made, there were fewer over the germinal vesicle than over any other part of the oocyte of comparable area (a ratio of about 3:5), excluding the zona pellucida. Radiographs were also prepared with granulosa cells, in which there is known to be an estradiol receptor (Richards, '751, for comparison to the radiographs of oocytes. It is readily apparent that many silver grains were concentrated over the nuclei when these cells were cultured under conditions identical to those employed with denuded oocytes. M,
DISCUSSION
Previous work has demonstrated that estradiol-17P reversibly inhibits the maturation of denuded porcine oocytes, and that progesterone will overcome this inhibitory effect (McGaughey, '77a). We have expanded this observation in the present study by determining that estradio:-17P, as compared with estradiol-17&,DES, progesterone, cortisol, and testosterone, is the most effective and consistent inhibitor of maturation for the porcine oocyte. In addition we have now demonstrated the following in respect to the inhibitory influence of estradiol-17P on porcine oocytes in vitro. (1)Inhibition by estradiol appears to be dependent upon t h e presence of a proteinaceous macromolecule (i.e., BSA in this study); particularly in the case of denuded oocytes. ( 2 ) Estradiol-17& at a sufficiently high concentration (i.e., 10 pg/ml), inhibits the maturation of intact porcine oocytes as well as denuded oocytes. (3) The nonsteroidal estrogen, DES, fails to inhibit maturation. (4) There is some stereospecificity involved since estradiol-17a does not inhibit overall maturation, but does inhibit the first meiotic division in denuded oocytes. A reasonable explanation for the different results obtained with media containing BSA
87
or dextran concerns the relative affinity of estradiol for these two macromolecules. We have observed that when BMOC-3 containing BSA and modified BMOC-3 containing dextran were incubated with 3H-estradiol (3.3 x M) and subsequently dialyzed against distilled water for 36 hours a t 4"C, 66% of the total estradiol was bound by the BSA of BMOC-3 while no steroid was bound by dextran (results not shown). This observation suggests that BSA might act in one of two ways to facilitate the inhibitory influence of estradiol on porcine oocytes. I t may increase the solubility of estradiol by binding it, which could result in a higher effective concentration of the steroid in BMOC-3 than in modified BMOC-3. BSA may also act as a carrier molecule to transport estradiol into the oocyte since i t is known that proteins including BSA can enter mammalian oocytes (Glass, '63, '71). In light of our association-dissociation studies (see below), the present results with denuded oocytes (cf. table 2 ) are more consistent with the latter explanation. At present, no clear explanation can be offered for the observation that cortisol may have inhibited the maturation (i.e., diakinesis to metaphase 2) of intact oocytes in modified BMOC-3 (cf. statistical caveat in RESULTS section). However, denuded oocytes were not inhibited by cortisol. The possibility therefore exists that cortisol may have interacted with the surrounding granulosa cells, in the case of intact oocytes, and brought about the observed inhibition indirectly. McNatty e t al. ('75) found t h a t fluid from small follicles contained as much as approximately 300 ng of estradiol per ml; while nearly 33 times this concentration (i.e., 10 p g per ml) was employed in our experiments to inhibit the maturation in vitro of porcine oocytes. In an earlier study (McGaughey, '77a), however, 1.0 p g of estradiol per ml also caused a statistically significant inhibition of maturation in denuded porcine oocytes in vitro. Currently it is not known what proportion of follicular fluid steroids are bound to macromolecules (Cook et al., '77; Mahajan and Little, '78), or what macromolecules might be required for the transport of steroids from one follicular compartment to another (Baird, '77; Moor, '77). In our present experiments in which estradiol (10.0 pg/ml) inhibited maturation in BMOC-3, the BSA concentration was only 4.5 mg per ml as compared with nearly ten times that concentration of albu-
88
JOEL D. RICHTER AND ROBERT W. MCGAUGHEY
min known to be present in porcine follicular fluids (McGaughey, '75). The possibility exists t h a t certain macromolecules of follicular fluid, particularly the nonspecific steroidbinding molecule albumin, might facilitate the interactions between steroids and cells, including the oocyte, of the follicle. Thus, although the concentration of estradiol presently employed to examine the inhibition of maturation in denuded porcine oocytes might be considered high as compared with steroid concentrations present in follicular fluid, i t should be borne in mind t h a t the minimal media, BMOC-3 with BSA or dextran, probably exhibit very little capacity for steroid binding and transport in comparison to antral fluids. In our experiments we observed that the uptake of 3H-estradiol by porcine oocytes reaches a plateau within two hours of culture when the steroid concentration is well below that found in the fluid of porcine follicles. The concentration of 3H-estradiol employed in M) was apthese experiments Le., 3.3 x proximately two orders of magnitude lower than has been reported for antral fluid of porcine follicles (Hunter et al., '76). Our data relating to the dissociation of 3H-estradiol show that the dissociation of this steroid is much slower in the presence of either radioinert estradiol or progesterone than is its initial association with denuded oocytes. The association of estradiol with denuded oocytes is quantitatively greater Le., 57% more estradiol uptake) in medium containing dextran as compared with medium containing BSA (cf. fig. 1).This observation may be related to the fact mentioned above that BSA binds estradiol significantly relative to dextran. It would appear that a certain amount of BSAbound estradiol may not be available for association with denuded oocytes. Even after a 3-hour culture in excess radioinert steroid, the vast majority of initially associated 3Hestradiol remains associated with the oocytes. This indicates that the oocytes have a very high capacity for steroids which is confirmed by data presented in figure 2. The same amount of labeled estradiol enters oocytes over a 1,000-fold range of excess unlabeled estradiol. The presence of large numbers of lipid vesicles (Szollosi, '72) would be expected to partition large amounts of steroids nonspecifically, which greatly complicates the search for estradiol receptors, if in fact they are present in oocytes.
We observed by autoradiography that in contrast to porcine cumulus granulosa cells which selectively accumulated radiolabeled estradiol within their nuclei, porcine oocytes did not exhibit selectively localized silver grains over their germinal vesicles. Cytological methods required in the autoradiographic experiments of the present study, particularly the dehydration steps needed for preparation of plastic-embedded oocytes, would be expected to result in the extraction of some of the associated 3H-estradiol. Therefore, the amounts of radiolabel observed in these studies would be expected to represent an underestimate of the total steroid associated with the oocytes. Whether it was unaltered estradiol-17P or some metabolite of this steroid which was associated with and inhibited the maturation of porcine oocytes in this study is not yet clear. In a different system, the induction of maturation in the amphibian oocyte by progesterone, it is known that these oocytes can metabolize steroids and that the metabolites of progesterone maintain their inductive activi t y (Reynhout and Smith, '73). The ability of porcine oocytes to metabolize steroids is not currently known. Our present results, therefore, cannot be interpreted as evidence for the presence of specific receptors for estradiol-17P in porcine oocytes. The nonsteroidal estrogen diethyl stilbestrol, which binds estradiol receptors in other systems (Ruh and Baudendistel, '771, failed to elicit an inhibitory effect on maturation. We have also cultured denuded oocytes in medium containing both estradiol-17P and the estrogen antagonist nafoxidine (Lazier and Alford, '77) in a n attempt to preclude inhibition. However, nafoxidine, a t equal or greater concentrations as estradiol, caused cytoplasmic shrinkage and aberrant chromosomal configurations. Furthermore, the large steroid capacity of the oocyte makes the search for low capacity, high affinity receptors extremely difficult. Our autoradiographic observations could be interpreted to suggest the possibility that estradiol might act on porcine oocytes at a cellular site other than the nucleus. In another system, it is known that steroids can alter the ion permeability of amphibian oocyte membranes (Morrill et al., '77; O'Connor et al., '77) and that this alteration leads to maturation (Baulieu et al., '78). It is also known that steroids can influence the expression of cyclic
STEROID INHIBITION OF PORCINE OOCYTE MATURATION
AMP (Greengard, '78). The ability of dibutyryl cyclic AMP to inhibit the maturation of mouse oocytes is well documented (Wassarman and Turner, '76; Stern and Wassarman, '74); and the possibility that estradiol may act on porcine oocytes indirectly by means of the intermediate, cyclic AMP, is currently being examined in our laboratory. Taken together, we feel that although the possibility of estradiol receptors does exist, our data indicate that the specific action of the steroid may be a t an extranuclear site, perhaps the membrane. Recently it was reported that estradiol-17P does not inhibit the maturation of mouse oocytes in vitro (Eppig and Koide, '78). We have also observed that estradiol- 17p, even at the very high concentration of 25 pg/ml, does not decrease the incidence of maturation in mouse oocytes (unpublished observations). At present, the difference in response to estradiol between mouse and porcine oocytes may be ascribed to several explanations. Mouse oocytes used for studies of maturation in vitro are collected from large, preovulatory follicles (Donahue, '68; Schultz and Wassarman, '771, whereas porcine oocytes routinely have been harvested from small ovarian follicles (McGaughey, '78). Mouse oocytes selected for culture are easily denuded of their loosely associated cumulus granulosa cells (Donahue, '681, whereas porcine oocytes from small follicles exhibit very tightly adherent cumulus granulosa and corona radiata cells which are anchored to the oocytes by means of cytoplasmic projections (McGaughey, '78). In addition, the mouse oocyte exhibits a very short germinal vesicle stage in culture (i.e., approximately 2 hours; Donahue, '68), while the germinal vesicle of the porcine oocyte remains intact for approximately 10 to 15 hours prior to the resumption of meiosis in vitro (McGaughey and Polge, '71; McGaughey, '78). It therefore appears probable that mouse oocytes and porcine oocytes might constitute rather different physiological models regarding follicular stage and the influence of steroids on maturation in vitro. Whether the maturation of mammalian oocytes in vitro (i.e., spontaneous maturation) represents a normal physiological process or a degenerative artificial phenomenon has been questioned (e.g., Eppig and Koide, '78). Although some apparently normal fetuses have been obtained from mouse oocytes which were matured in vitro (Cross and Brinster, '70),
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other studies have reported t h a t oocytes matured in vitro do not have normal capacity for further development (Niwa and Chang, '75; Moor and Trounson, '77). In contrast we recently demonstrated t h a t rabbit oocytes are capable of fertilization and preimplantation development in vivo following maturation in chemically defined medium in vitro, and that the patterns of polypeptide synthesis were comparable for rabbit oocytes matured in vivo and in vitro (Van Blerkom and McGaughey, '78a,b). Additional work is necessary to further assess the level of normalcy exhibited by mammalian oocytes matured in vitro, and to characterize the physiological requirements for maturation in vivo. The application of culture procedures currently appears to be necessary for the experimental dissection of the ovarian follicular components which very probably influence the maturation of mammalian oocytes. ACKNOWLEDGMENTS
The authors are grateful to Cudahy Foods of Phoenix, Arizona, for supplying the porcine reproductive tracts and to t h e Upjohn Company for supplying the nafoxidine. This study was supported in part by Grant HD06532 from the NIH and by the Arizona State University Research Enrichment Program. LITERATURE CITED Baird, D. T. 1977 Evidence in uiuo for the two-cell hypothesis of oestrogen synthesis by the sheep Graafian follicle. J. Reprod. Fert., 50: 183-185. Baulieu, E. E., F. Godeau, M. Schorderet and S. SchoderetSlatkin 1978 Steroid-induced meiotic division in Xenopus laeuis, oocytes: Surface and calcium. Nature, 275: 593. Brinster, R. L. 1971 Measuring embryonic enzyme activity. In: Methods in Mammalian Embryology. J. C. Daniel, Jr., ed. W. H. Freeman and Co., San Francisco, pp. 215-227. Cook, B., R. H. F. Hunter and A. S. Kelley 1977 Steroidbinding proteins in follicular fluid and peripheral plasma from pigs, cows and sheep. J. Reprod. Fert., 50: 65-71. Cross, P. C., and R. L. Brinster 1970 In uitro development of mouse oocytes. Biol. Reprod., 3: 298-307. Donahue, R. P. 1968 Maturation of the mouse oocyte in uitro. I. Sequence and timing of nuclear progression. J. Exp. Zool., 169: 237-250. Eppig, J. J., and S. L. Koide 1978 Effects of progesterone and oestradiol-17P on the spontaneous meiotic maturation of mouse oocytes. J. Reprod. Fert., 53: 99-101. Glass, L. 1963 Transfer of native and foreign serum antigens to oviducal mouse eggs. Am. Zool., 3: 135-156. 1971 Transmission of maternal protein into oocytes. In: Advances in the Biosciences 6. Schering Symposium on Intrinsic and Extrinsic Factors in Early Mammalian Development. G. Rasp& ed. Pergamon Press, New York, pp. 29-67.
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