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Morphologic and functional studies of immature rat oocyte-cumulus complexes after cryopreservation*†
FERTILITY AND STERILITY
Vol. 50, No. 5, November 1988
Copyright c 1988 The American Fertility Society
Printed in U.S.A .
Morphologic and functional studies of immature rat oocyte-cumulus complexes after cryopreservation*t Antonio Pellicer, M.D.:j:§ Abraham Lightman, M.D.:j: Toni G. Parmer, Ph.D.~
Harold R. Behrman, Ph.D.~ Alan H. De Cherney, M.D.:j:
Yale University School of Medicine, New Haven, Connecticut
The effects of freezing and thawing (F /T) on functional activity of immature rat oocyte-cumulus complexes (OCC) were studied. The OCC were divided into three groups according to the number of cumulus-cell layers surrounding them. The OCC were then frozen and thawed (F /T), with 'dimethyl sulfoxide (DMSO) as cryoprotectant. The survival rates after thawing increased significantly (P < 0.001) as the number of cumuluscell layers increased. Germinal vesicle breakdown (G VBD) was evaluated in F /T oocytes. After 2 hours, significantly (P < 0.05) fewer oocytes demonstrated GVBD than did those in the control group. There was no difference, however, after 4 hours of culture. A significant (P < 0.05) decrease in follicle-stimulating hormone (FSH)-dependent cyclic adenosine monophosphate (cAMP) accumulation was observed in the F/T group. However, the amount of cAMP produced was sufficient to maintain the oocyte in meiotic arrest. There was a borderline significant decrease of the coupling between cumulus cells and the oocyte in F /T OCC, as evaluated by the transport of 3H-uridine into the oocyte. It is concluded that immature rat oocytes can be successfully cryopreserved when they are surrounded by five or more layers of cumulus cells. FSH responsiveness and intercellular communication were essentially maintained. There was a slight delay in GVBD, which needs further clarification. Fertil Steril 50:805, 1988
The use of superovulation protocols in human in vitro fertilization and embryo transfer (IVF-ET), and gamete intrafallopian transfer (GIFT) results in multiple follicular development and, as a consequence, retrieval of multiple oocytes. When the established guidelines for human chorionic gonadoReceived September 21, 1987; revised and accepted July 25, 1988. *Supported by the National Institutes of Health grant HD10718, and by a Fulbright Fellowship to Dr. Pellicer. t Presented at the Forty-Third Annual Meeting of The American Fertility Society, September 28 to 30, 1987, Reno, Nevada. +Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology. §Present address and reprint requests: A. Pellicer, M.D., Department of Obstetrics and Gynecology, Hospital Clinico Universitario, Avda. Blasco Ibanez, 17, Valencia-46010, Spain. ~ Reproductive Biology Section, Departments of Obstetrics and Gynecology and Pharmacology.
tropin (hCG) injection and oocyte capture are followed, some of the retrieved oocytes are expected to be immature. These immature oocytes are surrounded by several layers of compact cumulus cells and characterized by the presence of a germinal vesicle in the cytoplasm. From animal 1 •2 and human3·4 studies, it can be concluded that immature oocytes cultured in vitro are able to mature, fertilize, and cleave. Between four and six oocytes are commonly used in the ongoing cycle in GIFT and IVF-ET procedures. The remaining oocytes, either mature or immature, can be successfully cryopreserved. 5 •6 It is generally accepted, however, that immature oocytes are less likely to survive freezing and thawing (F /T) than are mature ones. 7- 10 A possible method to increase the survival rate of immature oocytes after F /T is to procede with cryopreservation while maintaining the cumulus-
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cell layers that surround those oocytes. Evidence from studies carried out with mature oocytes, however, show that the presence of cumulus cells does not affect the survival rate of the F /T oocytes. 7·11 Data concerning a protective effect of the cumulus cells on the immature oocyte are unavailable at present. Although the precise role of the cumulus cells in F /T is uncertain, it has been established that cumulus cells are important for the development of the immature oocyte. More than 85% of the metabolites that enter the follicle-enclosed oocytes are originally taken up by the cumulus cells, and transferred into the oocyte via intercellular communication between both cellular compartments. 12 These transzonal junctional processes have been proposed to serve a nutritional role and to be responsible for oocyte growth during folliculogenesis. 13 Growth is critical for the oocyte's ability to resume meiosis. 14 It can also be achieved in vitro, but the follicular cells must remain in contact with the oocyte.15 There is also evidence supporting the fundamental role played by cyclic adenosine monophosphate (cAMP) in the inhibition of oocyte maturation. 16 Intraoocyte cAMP appears to be derived from the cumulus compartment, because elevation of cAMP levels in the oocyte-cumulus complex (OCC) is associated with an increase of cAMP levels within the oocyte. 17 The present study was conducted in order to evaluate the immature OCC after F/T. First, the possible role of cumulus cells on oocyte survival and the process of germinal vesicle breakdown (GVBD) were analyzed. Second, the function of the cumulus cells after F /T was tested by their ability to accumulate cAMP after FSH stimulation. Finally, the intercellular communication between oocyte and cumulus cells was evaluated with the use of the transport of 3H-uridine as a metabolic marker. MATERIALS AND METHODS Hormones, Drogs and Reagents
FSH (human FSH; NIADDK; AFP-4161 B; 3500 IU/mg; National Institutes of Health, Bethesda, MD) was diluted in Earle's minimal essential medium (MEM 2360, Gibco, Long Island, NY) containing 1 mg/ml bovine serum albumin (BSA) and 0.29 mg/ml glutamine. Dimethyl sulfoxide (DMSO) and unlabeled uri806
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dine were obtained from Sigma Chemical Co. (St. Louis, MO). 3H-uridine (40 Ci/mmol) was purchased from Amersham (Arlington Heights, IL). Retinoic acid was obtained from Sigma and dissolved in DMSO. Oocytes
Follicular development was stimulated in immature (24 to 27 days old) rats (crl:CD (SD) BR strain of Sprague-Dawley (Charles River Laboratories, Wilmington, MA) by subcutaneous injection of 10 IU pregnant mare serum gonadotropin (PMSG) (Gestil, Diosynth Inc., Chicago, IL). The animals were sacrificed 44 to 48 hours later. The largest follicles were punctured. Then, the OCC were isolated in MEM and either cryopreserved or incubated as a control at 37oC in a humidified 5% C02/95% air atmosphere. Freezing-Thawing Procedure
The technique used for F /T was based on the method described by Chen. 5 Before freezing, the OCC were transferred to dishes containing modified Dulbecco's phosphate-buffered salt solution (PB1) 7 supplemented with 10% fetal calf serum (FCS, Gibco) at room temperature. Between 10 and 20 OCC were placed in 0.1 ml of medium in sterile tissue culture glass ampules (Cryule, Wheaton Scientific, Millvill, NJ) and cooled on crushed ice to ooc. Subsequently, 0.1 ml of 3 M DMSO, previously cooled to ooc, was added to the ampules (final concentration 1.5 M DMSO). This mixture was then allowed to equilibrate for 15 minutes. After equilibration, the glass ampules were flame-sealed and placed in a programmable biological freezer (Planer R-204, Sunbury-on-Thames, Middlesex, England). The temperature was lowered to -7oC at a rate of 1 oC/minute. Seeding was induced by touching each ampule at the meniscus with a pair of forceps previously cooled in liquid nitrogen (LN 2). After being held for 10 minutes at the seeding temperature, the ampules were cooled to -36°C at a rate of 0.5°C/minute, and then plunged into LN 2 at -196oC for storage. The OCC were kept in LN 2 for a minimum of 3 days and a maximum of 2 weeks. Thawing was achieved by gently agitating the ampules in a water bath at 37oC until all traces of ice had disappeared. Subsequently, -0.8 ml of PB1 plus 10% FCS was added to the ampules at room temperature. Groups of 15 to 20 OCC were washed Fertility and Sterility
in PB1 and MEM and further cultured in 1 ml of the latter medium. Oocytes were considered to have survived F /T when they displayed a clear, bright, and homogeneous cytoplasm and an intact zona pellucida. There was virtually no space between the zona pellucida and the cytoplasm. Experiment 1: The Role of Cumulus Cells in the Survival of the Oocytes After F /T
To evaluate the role of the cumulus-cell layers in the survival of the oocytes after F /T, the OCC obtained from the preovulatory follicles were divided into three groups. Group A was composed of oocytes surrounded by five or more layers of cumulus cells. Group B was defined as oocytes surrounded by 2 or 3 layers of cumulus cells. These oocytes were obtained after pipetting through a narrow bore glass pipette. And group C was comprised of oocytes denuded by repeated pipetting. The survival rates were evaluated after F /T.
from each group were added to 12 X 75 mm glass culture tubes containing 0.5 ml medium with FSH (30 miU/ml). The OCC were incubated for 2 hours at 37oC in a shaking water bath under a humidified 5% C02/95% air atmosphere. After incubation, the culture tubes were immersed in a water bath (80°C) for 10 minutes and stored at -20oC for later analysis of cAMP by radioimmunoassay as described by Dorflinger et al. 19 In order to assess the biologic activity of cumulus cells after F /T, OCC previously subjected to F /T and their controls were cultured in medium containing FSH (30 miU/ml). Both control and F/T OCC were further divided into two subgroups and incubated for 1 or 2 hours. GVBD was evaluated after denuding the OCC. Experiment 4: Intercellular Communication Between the Oocyte and the Cumulus Cells After F /T
In a third experiment, the function of cumulus cells after F /T was evaluated by biochemical and biological parameters. The action ofFSH on cumulus cells was selected as a functional marker because FSH has a significant effect on stimulation of cAMP accumulation by the cumulus cells as well as in the production of a dose-dependent inhibition of oocyte maturation in the cumulus-enclosed oocyte.ts In order to evaluate the ability of cumulus cells to accumulate cAMP after FSH stimulation, OCC from F /T and the controls were cultured for 30 minutes in MEM. Then, groups of 20 to 30 OCC
Metabolic coupling was measured as an expression of intercellular communication between cumulus cells and oocytes. 13•20•21 The OCC from F /T and control groups were cultured for 30 minutes in MEM, and subsequently in the presence of 3H-uridine for 1 hour. Unlabeled uridine was mixed with 3 H-uridine before incubation in order to achieve a final concentration of 10 ~tCi/nmol. Addition of 10 ~tCi of this material produced a final concentration of 60 ~tM uridine in each incubation well. At the end of the incubation, half of the OCC were denuded as described above. The remaining OCC and the denuded oocytes were washed to remove the labeled uridine by sequential transfer through drops of medium. Groups of 10 oocytes and 10 OCC were transferred to separate scintillation vials, solubilized in 50 ~tl1 N NaOH, and neutralized with 60 ~tl HCl. Uridine uptake was measured by scintillation spectroscopy with Opti-Fluor (Packard Instrument Co., Downers Grove, IL). Intercellular communication (metabolic coupling) between the cumulus cells and the oocyte was expressed as the proportion of 3H-uridine found in the oocyte relative to total uptake in cumulus cells. 20 Retinoic acid (RA), an agent known to break intercellular communication between cells,22•23 was used as a positive control for complete uncoupling. Again, the OCC were first cultured for 30 minutes in MEM, and then in medium containing 3 H-uridine (60 ~tM) and RA (10-4 M). After incubation for 1 hours, the OCC were treated as described above, and the metabolic coupling was measured.
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Experiment 2: The Effect ofF/T on Spontaneous GVBD
In this experiment and all subsequent experiments, only OCC containing five or more cumuluscell layers were used. OCC were divided into two groups; one group was subjected to F /T and the other served as a control. OCC from the F /T and control groups were cultured in MEM plus BSA for 2 hours and 4 hours and the occurrence of G VBD observed. Time "zero" was considered the isolation from the follicles for the control OCC, and the thawing for the F /T OCC. The disappearance of the germinal vesicle was assessed by observation under Nomarski optics after denuding the oocytes. Experiment 3: The Function of Cumulus Cells After F /T
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Table 1 Survival Rates After F /T According to the Number of Cumulus Cells Surrounding the Oocytes: Results of Six Experiments
Group A" Bb
c
No.OCC frozen
No.OCC recovered after thawing
No. surviving oocytes
%
94 54 63
88 50 58
64 21 10
73 42 17
• Group A is significantly different from Band C (P < 0.001). b Group B is significantly different from C (P < 0.01).
Table 2 Percentage of Spontaneous G VBD in F /T and Control Oocytes After 2 and 4 Hours of Culture: Results of Four Experiments Group
2 Hours•
4 Hours•
Control F/T
77 (53)b 57 (74)
87 (54)• 83 (53)
• Number in parentheses represents total number of oocytes examined. b P < 0.05 control versus F /T at 2 hours. • P > 0.05 control versus F /Tat 4 hours.
The Function of the Cumulus Cells After F/T Statistical Analysis
The survival rate after F /T and the incidence of GVBD were compared between groups with the chi-square test. The effect ofFSH on cAMP levels was evaluated by Student's t-test. The difference in intercellular coupling was evaluated by the analysis of variance. Differences among groups were tested using Duncan's multiple range test. Values were expressed as the mean ± standard error of the mean (SEM). Differences between groups were considered statistically significant when P < 0.05.
RESULTS The Role of the Cumulus Cells in the Survival of the Oocytes After F /T
Table 1 shows the survival rates of the three groups of OCC according to the number of cumulus-celllayers that surrounded the oocyte at freezing. OCC containing five or more layers of cumulus cells (group A) showed a significantly (P < 0.001) higher survival rate than OCC with two or three cumulus layers (group B) and denuded oocytes (group C). Group B oocytes also survived significantly (P < 0.01) better than group C oocytes. The overall survival rate was 48.5%. The Effect ofF/Ton Spontaneous GVBD
The process of G VBD in OCC cultured in vitro was evaluated after 2 and 4 hours of culture. After 2 hours in culture, oocytes subjected to cryopreservation demonstrated a significant (P < 0.05) decrease in the percentage of spontaneous GVBD compared to the controls. However, after 4 hours in culture, the percentage of oocytes undergoing GVBD was similar in both groups (Table 2). 808
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The ability of FSH to stimulate cAMP accumulation and to prevent spontaneous GVBD was tested in separate experiments in the F /T and control OCC. FSH -dependent cAMP accumulation was significantly (P < 0.05) decreased in the F/T OCC compared to the controls after 1 hour (0.31 ± 0.04 versus 0.59 ± 0.04 pmol/OCC, respectively) and 2 hours (0.61 ± 0.07 versus 0.97 ± 0.07 pmol/ OCC, respectively) of culture (Table 3). In another set of four experiments, the ability of FSH to maintain the oocytes at the dictyate stage was evaluated. After 1 hour, the percentage of GVBD was 9.3% compared to 20.6% for the controls (not significant [NS]). After 2 hours, the percentages of GVBD were 25.9% and 56.8%, respectively (NS). The percentage of GVBD in the F/T oocytes after 2 hours in the presence of FSH (25.9%) was significantly (P < 0.001) decreased in comparison to the spontaneous GVBD after 2 hours observed in experiment 2 (57%). Similarly, FSH significantly (P < 0.05) reduced the percentage ofGVBD in the controls from 77% (experiment 2) to 56.8%. Therefore, FSH was effective in preTable 3 FSH-Dependent cAMP Accumulation (pmolfOCC) and Percentage of GVBD. Each Parameter Was Evaluated in Four Different Experiments Control Culture time (hours)
GVBD
cAMP (pmol/OCC) Mean± SEM
%
1 2
20.6 (34)" 56.8 (74)
F/T
GVBD
cAMP (pmol/OCC) Mean±SEM
%
0.59 ± 0.04b o.97 ± o.o1•
9.3 (43) 25.9 (27)
0.31 ± 0.04 0.61 ± 0.07
• Number in parentheses represents total number ofOCC examined. b P < 0.05 control versus F /T at 1 hour. • P < 0.05 control versus F /Tat 2 hours.
Fertility and Sterility
Table 4 Uptake of 3 H-uridine {fmol) by the OCC and Oocyte, and Measurements of the Metabolic Coupling {MC). Results of Four Experiments Group
fmol/oocyte
fmol/OCC
MC
Control
145.6 ± 7.8 111.5 ± 10.8 35.5 ± 13.1
999.1 ± 194.2 1125 ± 169.1 2799 ± 367.3
16.7 ± 2.0" 10.6 ± 1.4 1.3 ± 0.5b
F/T RA •P
=
0.057 control versus F /T.
bRA + control and F /T.
venting spontaneous GVBD in the F/T OCC as well as in the controls. Intercellular Communication Between the Cumulus Cells and the Oocyte After F/T
Metabolic cooperativity between cumulus cells and the oocyte was assessed by analysis of 3 H-uridine uptake. OCC subjected to F /T showed a borderline significant reduction in coupling compared to the controls (10.6 ± 1.4 versus 16.7 ± 2.0, respectively, P = 0.057). However, when compared to uncoupling induced by RA, both F /T and control groups showed a significantly (P < 0.001) higher coupling index (Table 4).
however, both groups showed similar rates of GVBD. This initial delay in GVBD probably reflects a slow reinitiation of all metabolic functions arrested during cryopreservation. However, as there is some concern regarding the effect of cryopreservation on spindle formation, 24 the present findings require further studies on other phenomena related to oocyte maturation. The presence of FSH in the culture medium delayed spontaneous GVBD in the F/T oocytes. While the percentage of spontaneous GVBD after 2 hours was 57%, FSH significantly reduced GVBD to 26%. These results suggest that the amount of cAMP produced by the cumulus cells and transferred into the oocytes was sufficient to maintain the oocyte arrested in the dictyate stage, since cAMP is an important inhibitor of oocyte maturation.17,1s
This study shows that immature rat oocytes cryopreserved at -196oC were able to survive F /T and undergo GVBD. The overall survival rate was 48.5%. Survival rate was directly related to the number of cumulus-cell layers surrounding the oocyte. The survival rate was over 70% when 5 or more layers of cumulus cells were present. This rate was higher than what has previously been reported for immature rat oocytes8 and other species, including human oocytes. 7- 10 Denuded oocytes, showed only a 17% survival rate, which is similar to the rate reported in mice. 7 Thus, it appears that the cumulus layers exert an important protective effect on the immature oocyte subjected to cryopreservation. This is in contrast to the mature oocyte, where the presence of cumulus cells does not affect9 •11 the survival rate. It should be also pointed out, however, that oocytes in group C were obtained after repeated pipetting. This could potentially damage the oocytes and reduce their survival rates after F /T. The process of spontaneous GVBD after 2 hours of incubation was significantly delayed in the F /T oocytes as compared to the controls. After 4 hours,
The decrease obtained in FSH-induced cAMP accumulation by the F /T OCC, could be due to the presence of DMSO in the cells, since we have observed a decrease in cAMP accumulation by OCC incubated with DMS0. 25 This finding could also be explained by the degeneration of some of the cumuIus cells after F /T, which reduced the ability of the OCC to accumulate cAMP. Nevertheless, the FSH-dependent cAMP accumulation in the cumulus cells was sufficient to maintain the oocytes in the arrested stage. Thus, we conclude that the cumulus cells remained functional after F /T. In order to ascertain the degree of intercellular communication, studies with radioactive uridine were carried out. Uridine has previously been shown to be a good marker of metabolic coupling between cumulus cells and oocyte in mammals. 20·21 The results of our study show that there was a borderline significant decrease in metabolic coupling after F /T. RA was also examined because of its established property of inducing disruption of the intercellular communication in other cellular systems. The comparison between the metabolic coupling in the F/T and control OCC to the RAtreated OCC clearly showed that there was metabolic cooperation after F /T in the rat oocyte. In summary, the present studies demonstrate that immature rat oocytes can be successfully cryopreserved when they are surrounded by five or more layers of cumulus cells. FSH responsiveness and intercellular communication are essentially maintained. There was a slight delayinG VBD. Additional information is needed on the ability of immature oocytes to undergo complete maturation, fertilization, cleavage, and further development be-
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fore they can be considered as an option in IVF-ET and GIFT procedures. 13. REFERENCES 1. Pincus G, Enzmann EV: The comparative behaviour of mammalian eggs in vivo and in vitro. I. The activation of mammalian eggs. J Exp Med 62:665, 1935 2. Chang MC: The maturation of rabbit oocytes in culture and their maturation, activation, fertilization, and subsequent development in the fallopian tube. J Exp Zool128:379, 1955 3. Edwards RG, Bavister BD, Steptoe PC: Early stages offertilization in vitro of human oocytes matured in vitro. Nature 221:632, 1969 4. Veeck LL, Wortham JWE Jr, Witmyer J, Sandow BA, Acosta AA, Garcia JE, Jones GS, Jones HW Jr: Maturation and fertilization of morphologically immature human oocytes in a program of in vitro fertilization. Fertil Steril 39: 594,1983 5. Chen C: Pregnancy after human oocyte cryopreservation. Lancet 1:884, 1986 6. Chen C: Pregnancies after human oocyte cryopreservation. Presented at the Vth World Congress on In Vitro Fertilization and Embryo Transfer, Norfolk, Virginia, AprilS to 10, 1987. Published by the American Fertility Society, Birmingham, Alabama, p 19 7. Whittingham DG: Fertilization in vitro and development to term of unfertilized mouse oocytes previously stored at -19s•c. J Reprod Fertil49:89, 1977 8. Kasai M, Iritani A, Chang MC: Fertilization in vitro of rat ovarian oocytes after freezing and thawing. Biol Reprod 21: 839,1979 9. Al-Hasani S, Tolksdorf A, Diedrich K, Van der Ven H, Krebs D: Successful in-vitro fertilization of frozen-thawed rabbit oocytes. Hum Reprod 1:309, 1986 10. Mandelbaum J, Junca AM, Tibi C, Plachot M, Alnot MO, Salat-Baroux J, Cohen J: Cryopreservation of immature and mature human oocytes. Presented at the Vth World Congress of In Vitro Fertilization and Embryo Transfer, Norfolk, Virginia, April 5 to 10, 1987. Published by the American Fertility Society, Birmingham, Alabama, p 20 11. Parkening TA, Tsunoda Y, Chang MC: Effects of various low temperatures, cryoprotective agents and cooling rates on the survival, fertilizability and development of frozenthawed mouse eggs. J Exp Zool197:369, 1976 12. Heller DT, Cahill DM, Schultz RM: Biochemical studies
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14.
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of mammalian oogenesis: metabolic cooperativity between granulosa cells and growing mouse oocytes. Dev Biol 84: 455, 1981 Brower PT, Schultz RM: Intercellular communication between granulosa cells and mouse oocytes: existence and possible nutritional role during oocyte growth. Dev Biol 90: 144,1982 Ayalon D, Tsafriri A, Lindner HR, Cordova T, Harell A: Serum gonadotrophin levels in pro-estrous rats in relation to the resumption of meiosis by the oocyte. J Rep rod Fertil 31:51, 1972 Bachvarova R, Baran MM, Tejblum A: Development of naked growing mouse oocytes in vitro. J Exp Zool 211:159, 1980 Magnusson C, Hillensjo T: Inhibition of maturation and metabolism of rat oocytes by cyclic AMP. J Exp Zool 201: 139,1977 Bornslaeger EA, Schultz RM: Regulation of mouse oocyte maturation: effect of elevating cumulus cell cAMP on oocyte cAMP levels. Biol Reprod 33:698, 1985 Laufer N, Polan ML, DeCherney AH, Halsentine FP, Behrman HR: Characteristics of pre- and postovulatory oocyte cumulus complex responses to gonadotropins and adenosine. (Abstr.) Presented at the 65th Annual Meeting ofthe Endocrine Society, San Antonio, Texas, June 8 to 10, 1983,p300 Dorfingler LJ, Albert PJ, Williams AT, Behrman HR: Calcium is an inhibitor of luteinizing hormone-sensitive adenylate cyclase in the luteal cell. Endocrinology 114:1208, 1984 Salustri A, Siracusa G: Metabolic coupling, cumulus expansion and meiotic resumption in mouse cumuli oophori cultured in vitro in the presence ofFSH or dcAMP, or stimulated in vivo by hCG. J Reprod Fertil68:335, 1983 Moor RM, Polge C, Willadsen SM: Effect of follicular steroids on the maturation and fertilization of mammalian oocytes. J Embryol Exp Morphol56:319, 1980 Mehta PP, Bertram JS, Loewenstein WR: Growth inhibition of transformed cells correlates with their junctional communication with normal cells. Cell44:187, 1986 Wiilder L, Liitzelschwab R: Effects of 12-0-tetradecanoylphorbol-13-acetate (TPA), retinoic acid and diazepam on intercellular communication in a monolayer ofrat liver epithelial cells. Exp Cell Res 152:66, 1984 Magistrini M, Szollosi D: Effects of cold and of isopropylN-phenyl carbamate on the second meiotic spindle of mouse oocytes. Eur J Cell Biol 22:699, 1980 Pellicer A, Parmer TG, Behrman HR: Unpublished data.
Fertility and Sterility
Vol. 50, No. 5, November 1988
Copyright c 1988 The American Fertility Society
Printed in U.S.A .
Morphologic and functional studies of immature rat oocyte-cumulus complexes after cryopreservation*t Antonio Pellicer, M.D.:j:§ Abraham Lightman, M.D.:j: Toni G. Parmer, Ph.D.~
Harold R. Behrman, Ph.D.~ Alan H. De Cherney, M.D.:j:
Yale University School of Medicine, New Haven, Connecticut
The effects of freezing and thawing (F /T) on functional activity of immature rat oocyte-cumulus complexes (OCC) were studied. The OCC were divided into three groups according to the number of cumulus-cell layers surrounding them. The OCC were then frozen and thawed (F /T), with 'dimethyl sulfoxide (DMSO) as cryoprotectant. The survival rates after thawing increased significantly (P < 0.001) as the number of cumuluscell layers increased. Germinal vesicle breakdown (G VBD) was evaluated in F /T oocytes. After 2 hours, significantly (P < 0.05) fewer oocytes demonstrated GVBD than did those in the control group. There was no difference, however, after 4 hours of culture. A significant (P < 0.05) decrease in follicle-stimulating hormone (FSH)-dependent cyclic adenosine monophosphate (cAMP) accumulation was observed in the F/T group. However, the amount of cAMP produced was sufficient to maintain the oocyte in meiotic arrest. There was a borderline significant decrease of the coupling between cumulus cells and the oocyte in F /T OCC, as evaluated by the transport of 3H-uridine into the oocyte. It is concluded that immature rat oocytes can be successfully cryopreserved when they are surrounded by five or more layers of cumulus cells. FSH responsiveness and intercellular communication were essentially maintained. There was a slight delay in GVBD, which needs further clarification. Fertil Steril 50:805, 1988
The use of superovulation protocols in human in vitro fertilization and embryo transfer (IVF-ET), and gamete intrafallopian transfer (GIFT) results in multiple follicular development and, as a consequence, retrieval of multiple oocytes. When the established guidelines for human chorionic gonadoReceived September 21, 1987; revised and accepted July 25, 1988. *Supported by the National Institutes of Health grant HD10718, and by a Fulbright Fellowship to Dr. Pellicer. t Presented at the Forty-Third Annual Meeting of The American Fertility Society, September 28 to 30, 1987, Reno, Nevada. +Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology. §Present address and reprint requests: A. Pellicer, M.D., Department of Obstetrics and Gynecology, Hospital Clinico Universitario, Avda. Blasco Ibanez, 17, Valencia-46010, Spain. ~ Reproductive Biology Section, Departments of Obstetrics and Gynecology and Pharmacology.
tropin (hCG) injection and oocyte capture are followed, some of the retrieved oocytes are expected to be immature. These immature oocytes are surrounded by several layers of compact cumulus cells and characterized by the presence of a germinal vesicle in the cytoplasm. From animal 1 •2 and human3·4 studies, it can be concluded that immature oocytes cultured in vitro are able to mature, fertilize, and cleave. Between four and six oocytes are commonly used in the ongoing cycle in GIFT and IVF-ET procedures. The remaining oocytes, either mature or immature, can be successfully cryopreserved. 5 •6 It is generally accepted, however, that immature oocytes are less likely to survive freezing and thawing (F /T) than are mature ones. 7- 10 A possible method to increase the survival rate of immature oocytes after F /T is to procede with cryopreservation while maintaining the cumulus-
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805
cell layers that surround those oocytes. Evidence from studies carried out with mature oocytes, however, show that the presence of cumulus cells does not affect the survival rate of the F /T oocytes. 7·11 Data concerning a protective effect of the cumulus cells on the immature oocyte are unavailable at present. Although the precise role of the cumulus cells in F /T is uncertain, it has been established that cumulus cells are important for the development of the immature oocyte. More than 85% of the metabolites that enter the follicle-enclosed oocytes are originally taken up by the cumulus cells, and transferred into the oocyte via intercellular communication between both cellular compartments. 12 These transzonal junctional processes have been proposed to serve a nutritional role and to be responsible for oocyte growth during folliculogenesis. 13 Growth is critical for the oocyte's ability to resume meiosis. 14 It can also be achieved in vitro, but the follicular cells must remain in contact with the oocyte.15 There is also evidence supporting the fundamental role played by cyclic adenosine monophosphate (cAMP) in the inhibition of oocyte maturation. 16 Intraoocyte cAMP appears to be derived from the cumulus compartment, because elevation of cAMP levels in the oocyte-cumulus complex (OCC) is associated with an increase of cAMP levels within the oocyte. 17 The present study was conducted in order to evaluate the immature OCC after F/T. First, the possible role of cumulus cells on oocyte survival and the process of germinal vesicle breakdown (GVBD) were analyzed. Second, the function of the cumulus cells after F /T was tested by their ability to accumulate cAMP after FSH stimulation. Finally, the intercellular communication between oocyte and cumulus cells was evaluated with the use of the transport of 3H-uridine as a metabolic marker. MATERIALS AND METHODS Hormones, Drogs and Reagents
FSH (human FSH; NIADDK; AFP-4161 B; 3500 IU/mg; National Institutes of Health, Bethesda, MD) was diluted in Earle's minimal essential medium (MEM 2360, Gibco, Long Island, NY) containing 1 mg/ml bovine serum albumin (BSA) and 0.29 mg/ml glutamine. Dimethyl sulfoxide (DMSO) and unlabeled uri806
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dine were obtained from Sigma Chemical Co. (St. Louis, MO). 3H-uridine (40 Ci/mmol) was purchased from Amersham (Arlington Heights, IL). Retinoic acid was obtained from Sigma and dissolved in DMSO. Oocytes
Follicular development was stimulated in immature (24 to 27 days old) rats (crl:CD (SD) BR strain of Sprague-Dawley (Charles River Laboratories, Wilmington, MA) by subcutaneous injection of 10 IU pregnant mare serum gonadotropin (PMSG) (Gestil, Diosynth Inc., Chicago, IL). The animals were sacrificed 44 to 48 hours later. The largest follicles were punctured. Then, the OCC were isolated in MEM and either cryopreserved or incubated as a control at 37oC in a humidified 5% C02/95% air atmosphere. Freezing-Thawing Procedure
The technique used for F /T was based on the method described by Chen. 5 Before freezing, the OCC were transferred to dishes containing modified Dulbecco's phosphate-buffered salt solution (PB1) 7 supplemented with 10% fetal calf serum (FCS, Gibco) at room temperature. Between 10 and 20 OCC were placed in 0.1 ml of medium in sterile tissue culture glass ampules (Cryule, Wheaton Scientific, Millvill, NJ) and cooled on crushed ice to ooc. Subsequently, 0.1 ml of 3 M DMSO, previously cooled to ooc, was added to the ampules (final concentration 1.5 M DMSO). This mixture was then allowed to equilibrate for 15 minutes. After equilibration, the glass ampules were flame-sealed and placed in a programmable biological freezer (Planer R-204, Sunbury-on-Thames, Middlesex, England). The temperature was lowered to -7oC at a rate of 1 oC/minute. Seeding was induced by touching each ampule at the meniscus with a pair of forceps previously cooled in liquid nitrogen (LN 2). After being held for 10 minutes at the seeding temperature, the ampules were cooled to -36°C at a rate of 0.5°C/minute, and then plunged into LN 2 at -196oC for storage. The OCC were kept in LN 2 for a minimum of 3 days and a maximum of 2 weeks. Thawing was achieved by gently agitating the ampules in a water bath at 37oC until all traces of ice had disappeared. Subsequently, -0.8 ml of PB1 plus 10% FCS was added to the ampules at room temperature. Groups of 15 to 20 OCC were washed Fertility and Sterility
in PB1 and MEM and further cultured in 1 ml of the latter medium. Oocytes were considered to have survived F /T when they displayed a clear, bright, and homogeneous cytoplasm and an intact zona pellucida. There was virtually no space between the zona pellucida and the cytoplasm. Experiment 1: The Role of Cumulus Cells in the Survival of the Oocytes After F /T
To evaluate the role of the cumulus-cell layers in the survival of the oocytes after F /T, the OCC obtained from the preovulatory follicles were divided into three groups. Group A was composed of oocytes surrounded by five or more layers of cumulus cells. Group B was defined as oocytes surrounded by 2 or 3 layers of cumulus cells. These oocytes were obtained after pipetting through a narrow bore glass pipette. And group C was comprised of oocytes denuded by repeated pipetting. The survival rates were evaluated after F /T.
from each group were added to 12 X 75 mm glass culture tubes containing 0.5 ml medium with FSH (30 miU/ml). The OCC were incubated for 2 hours at 37oC in a shaking water bath under a humidified 5% C02/95% air atmosphere. After incubation, the culture tubes were immersed in a water bath (80°C) for 10 minutes and stored at -20oC for later analysis of cAMP by radioimmunoassay as described by Dorflinger et al. 19 In order to assess the biologic activity of cumulus cells after F /T, OCC previously subjected to F /T and their controls were cultured in medium containing FSH (30 miU/ml). Both control and F/T OCC were further divided into two subgroups and incubated for 1 or 2 hours. GVBD was evaluated after denuding the OCC. Experiment 4: Intercellular Communication Between the Oocyte and the Cumulus Cells After F /T
In a third experiment, the function of cumulus cells after F /T was evaluated by biochemical and biological parameters. The action ofFSH on cumulus cells was selected as a functional marker because FSH has a significant effect on stimulation of cAMP accumulation by the cumulus cells as well as in the production of a dose-dependent inhibition of oocyte maturation in the cumulus-enclosed oocyte.ts In order to evaluate the ability of cumulus cells to accumulate cAMP after FSH stimulation, OCC from F /T and the controls were cultured for 30 minutes in MEM. Then, groups of 20 to 30 OCC
Metabolic coupling was measured as an expression of intercellular communication between cumulus cells and oocytes. 13•20•21 The OCC from F /T and control groups were cultured for 30 minutes in MEM, and subsequently in the presence of 3H-uridine for 1 hour. Unlabeled uridine was mixed with 3 H-uridine before incubation in order to achieve a final concentration of 10 ~tCi/nmol. Addition of 10 ~tCi of this material produced a final concentration of 60 ~tM uridine in each incubation well. At the end of the incubation, half of the OCC were denuded as described above. The remaining OCC and the denuded oocytes were washed to remove the labeled uridine by sequential transfer through drops of medium. Groups of 10 oocytes and 10 OCC were transferred to separate scintillation vials, solubilized in 50 ~tl1 N NaOH, and neutralized with 60 ~tl HCl. Uridine uptake was measured by scintillation spectroscopy with Opti-Fluor (Packard Instrument Co., Downers Grove, IL). Intercellular communication (metabolic coupling) between the cumulus cells and the oocyte was expressed as the proportion of 3H-uridine found in the oocyte relative to total uptake in cumulus cells. 20 Retinoic acid (RA), an agent known to break intercellular communication between cells,22•23 was used as a positive control for complete uncoupling. Again, the OCC were first cultured for 30 minutes in MEM, and then in medium containing 3 H-uridine (60 ~tM) and RA (10-4 M). After incubation for 1 hours, the OCC were treated as described above, and the metabolic coupling was measured.
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Experiment 2: The Effect ofF/T on Spontaneous GVBD
In this experiment and all subsequent experiments, only OCC containing five or more cumuluscell layers were used. OCC were divided into two groups; one group was subjected to F /T and the other served as a control. OCC from the F /T and control groups were cultured in MEM plus BSA for 2 hours and 4 hours and the occurrence of G VBD observed. Time "zero" was considered the isolation from the follicles for the control OCC, and the thawing for the F /T OCC. The disappearance of the germinal vesicle was assessed by observation under Nomarski optics after denuding the oocytes. Experiment 3: The Function of Cumulus Cells After F /T
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Table 1 Survival Rates After F /T According to the Number of Cumulus Cells Surrounding the Oocytes: Results of Six Experiments
Group A" Bb
c
No.OCC frozen
No.OCC recovered after thawing
No. surviving oocytes
%
94 54 63
88 50 58
64 21 10
73 42 17
• Group A is significantly different from Band C (P < 0.001). b Group B is significantly different from C (P < 0.01).
Table 2 Percentage of Spontaneous G VBD in F /T and Control Oocytes After 2 and 4 Hours of Culture: Results of Four Experiments Group
2 Hours•
4 Hours•
Control F/T
77 (53)b 57 (74)
87 (54)• 83 (53)
• Number in parentheses represents total number of oocytes examined. b P < 0.05 control versus F /T at 2 hours. • P > 0.05 control versus F /Tat 4 hours.
The Function of the Cumulus Cells After F/T Statistical Analysis
The survival rate after F /T and the incidence of GVBD were compared between groups with the chi-square test. The effect ofFSH on cAMP levels was evaluated by Student's t-test. The difference in intercellular coupling was evaluated by the analysis of variance. Differences among groups were tested using Duncan's multiple range test. Values were expressed as the mean ± standard error of the mean (SEM). Differences between groups were considered statistically significant when P < 0.05.
RESULTS The Role of the Cumulus Cells in the Survival of the Oocytes After F /T
Table 1 shows the survival rates of the three groups of OCC according to the number of cumulus-celllayers that surrounded the oocyte at freezing. OCC containing five or more layers of cumulus cells (group A) showed a significantly (P < 0.001) higher survival rate than OCC with two or three cumulus layers (group B) and denuded oocytes (group C). Group B oocytes also survived significantly (P < 0.01) better than group C oocytes. The overall survival rate was 48.5%. The Effect ofF/Ton Spontaneous GVBD
The process of G VBD in OCC cultured in vitro was evaluated after 2 and 4 hours of culture. After 2 hours in culture, oocytes subjected to cryopreservation demonstrated a significant (P < 0.05) decrease in the percentage of spontaneous GVBD compared to the controls. However, after 4 hours in culture, the percentage of oocytes undergoing GVBD was similar in both groups (Table 2). 808
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Cryopreservation of immature oocytes
The ability of FSH to stimulate cAMP accumulation and to prevent spontaneous GVBD was tested in separate experiments in the F /T and control OCC. FSH -dependent cAMP accumulation was significantly (P < 0.05) decreased in the F/T OCC compared to the controls after 1 hour (0.31 ± 0.04 versus 0.59 ± 0.04 pmol/OCC, respectively) and 2 hours (0.61 ± 0.07 versus 0.97 ± 0.07 pmol/ OCC, respectively) of culture (Table 3). In another set of four experiments, the ability of FSH to maintain the oocytes at the dictyate stage was evaluated. After 1 hour, the percentage of GVBD was 9.3% compared to 20.6% for the controls (not significant [NS]). After 2 hours, the percentages of GVBD were 25.9% and 56.8%, respectively (NS). The percentage of GVBD in the F/T oocytes after 2 hours in the presence of FSH (25.9%) was significantly (P < 0.001) decreased in comparison to the spontaneous GVBD after 2 hours observed in experiment 2 (57%). Similarly, FSH significantly (P < 0.05) reduced the percentage ofGVBD in the controls from 77% (experiment 2) to 56.8%. Therefore, FSH was effective in preTable 3 FSH-Dependent cAMP Accumulation (pmolfOCC) and Percentage of GVBD. Each Parameter Was Evaluated in Four Different Experiments Control Culture time (hours)
GVBD
cAMP (pmol/OCC) Mean± SEM
%
1 2
20.6 (34)" 56.8 (74)
F/T
GVBD
cAMP (pmol/OCC) Mean±SEM
%
0.59 ± 0.04b o.97 ± o.o1•
9.3 (43) 25.9 (27)
0.31 ± 0.04 0.61 ± 0.07
• Number in parentheses represents total number ofOCC examined. b P < 0.05 control versus F /T at 1 hour. • P < 0.05 control versus F /Tat 2 hours.
Fertility and Sterility
Table 4 Uptake of 3 H-uridine {fmol) by the OCC and Oocyte, and Measurements of the Metabolic Coupling {MC). Results of Four Experiments Group
fmol/oocyte
fmol/OCC
MC
Control
145.6 ± 7.8 111.5 ± 10.8 35.5 ± 13.1
999.1 ± 194.2 1125 ± 169.1 2799 ± 367.3
16.7 ± 2.0" 10.6 ± 1.4 1.3 ± 0.5b
F/T RA •P
=
0.057 control versus F /T.
bRA + control and F /T.
venting spontaneous GVBD in the F/T OCC as well as in the controls. Intercellular Communication Between the Cumulus Cells and the Oocyte After F/T
Metabolic cooperativity between cumulus cells and the oocyte was assessed by analysis of 3 H-uridine uptake. OCC subjected to F /T showed a borderline significant reduction in coupling compared to the controls (10.6 ± 1.4 versus 16.7 ± 2.0, respectively, P = 0.057). However, when compared to uncoupling induced by RA, both F /T and control groups showed a significantly (P < 0.001) higher coupling index (Table 4).
however, both groups showed similar rates of GVBD. This initial delay in GVBD probably reflects a slow reinitiation of all metabolic functions arrested during cryopreservation. However, as there is some concern regarding the effect of cryopreservation on spindle formation, 24 the present findings require further studies on other phenomena related to oocyte maturation. The presence of FSH in the culture medium delayed spontaneous GVBD in the F/T oocytes. While the percentage of spontaneous GVBD after 2 hours was 57%, FSH significantly reduced GVBD to 26%. These results suggest that the amount of cAMP produced by the cumulus cells and transferred into the oocytes was sufficient to maintain the oocyte arrested in the dictyate stage, since cAMP is an important inhibitor of oocyte maturation.17,1s
This study shows that immature rat oocytes cryopreserved at -196oC were able to survive F /T and undergo GVBD. The overall survival rate was 48.5%. Survival rate was directly related to the number of cumulus-cell layers surrounding the oocyte. The survival rate was over 70% when 5 or more layers of cumulus cells were present. This rate was higher than what has previously been reported for immature rat oocytes8 and other species, including human oocytes. 7- 10 Denuded oocytes, showed only a 17% survival rate, which is similar to the rate reported in mice. 7 Thus, it appears that the cumulus layers exert an important protective effect on the immature oocyte subjected to cryopreservation. This is in contrast to the mature oocyte, where the presence of cumulus cells does not affect9 •11 the survival rate. It should be also pointed out, however, that oocytes in group C were obtained after repeated pipetting. This could potentially damage the oocytes and reduce their survival rates after F /T. The process of spontaneous GVBD after 2 hours of incubation was significantly delayed in the F /T oocytes as compared to the controls. After 4 hours,
The decrease obtained in FSH-induced cAMP accumulation by the F /T OCC, could be due to the presence of DMSO in the cells, since we have observed a decrease in cAMP accumulation by OCC incubated with DMS0. 25 This finding could also be explained by the degeneration of some of the cumuIus cells after F /T, which reduced the ability of the OCC to accumulate cAMP. Nevertheless, the FSH-dependent cAMP accumulation in the cumulus cells was sufficient to maintain the oocytes in the arrested stage. Thus, we conclude that the cumulus cells remained functional after F /T. In order to ascertain the degree of intercellular communication, studies with radioactive uridine were carried out. Uridine has previously been shown to be a good marker of metabolic coupling between cumulus cells and oocyte in mammals. 20·21 The results of our study show that there was a borderline significant decrease in metabolic coupling after F /T. RA was also examined because of its established property of inducing disruption of the intercellular communication in other cellular systems. The comparison between the metabolic coupling in the F/T and control OCC to the RAtreated OCC clearly showed that there was metabolic cooperation after F /T in the rat oocyte. In summary, the present studies demonstrate that immature rat oocytes can be successfully cryopreserved when they are surrounded by five or more layers of cumulus cells. FSH responsiveness and intercellular communication are essentially maintained. There was a slight delayinG VBD. Additional information is needed on the ability of immature oocytes to undergo complete maturation, fertilization, cleavage, and further development be-
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DISCUSSION
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fore they can be considered as an option in IVF-ET and GIFT procedures. 13. REFERENCES 1. Pincus G, Enzmann EV: The comparative behaviour of mammalian eggs in vivo and in vitro. I. The activation of mammalian eggs. J Exp Med 62:665, 1935 2. Chang MC: The maturation of rabbit oocytes in culture and their maturation, activation, fertilization, and subsequent development in the fallopian tube. J Exp Zool128:379, 1955 3. Edwards RG, Bavister BD, Steptoe PC: Early stages offertilization in vitro of human oocytes matured in vitro. Nature 221:632, 1969 4. Veeck LL, Wortham JWE Jr, Witmyer J, Sandow BA, Acosta AA, Garcia JE, Jones GS, Jones HW Jr: Maturation and fertilization of morphologically immature human oocytes in a program of in vitro fertilization. Fertil Steril 39: 594,1983 5. Chen C: Pregnancy after human oocyte cryopreservation. Lancet 1:884, 1986 6. Chen C: Pregnancies after human oocyte cryopreservation. Presented at the Vth World Congress on In Vitro Fertilization and Embryo Transfer, Norfolk, Virginia, AprilS to 10, 1987. Published by the American Fertility Society, Birmingham, Alabama, p 19 7. Whittingham DG: Fertilization in vitro and development to term of unfertilized mouse oocytes previously stored at -19s•c. J Reprod Fertil49:89, 1977 8. Kasai M, Iritani A, Chang MC: Fertilization in vitro of rat ovarian oocytes after freezing and thawing. Biol Reprod 21: 839,1979 9. Al-Hasani S, Tolksdorf A, Diedrich K, Van der Ven H, Krebs D: Successful in-vitro fertilization of frozen-thawed rabbit oocytes. Hum Reprod 1:309, 1986 10. Mandelbaum J, Junca AM, Tibi C, Plachot M, Alnot MO, Salat-Baroux J, Cohen J: Cryopreservation of immature and mature human oocytes. Presented at the Vth World Congress of In Vitro Fertilization and Embryo Transfer, Norfolk, Virginia, April 5 to 10, 1987. Published by the American Fertility Society, Birmingham, Alabama, p 20 11. Parkening TA, Tsunoda Y, Chang MC: Effects of various low temperatures, cryoprotective agents and cooling rates on the survival, fertilizability and development of frozenthawed mouse eggs. J Exp Zool197:369, 1976 12. Heller DT, Cahill DM, Schultz RM: Biochemical studies
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