Brief report Synthesis of ribonucleic acid in oocytes collected from squirrel monkeys and humans following chorionic gonadotropin administration.код для вставкиСкачать
American Journal of Primatology 5:267-270 (1983) BRIEF REPORT Synthesis of Ribonucleic Acid in Oocytes Collected From Squirrel Monkeys and Humans Following Chorionic Gonadotropin Administration K.J. HUTZ', G.B. HOLZMAN', A N U W.R. DUKELOW' 'Endocrine Research Unit and 'Departme& of Obstetrics and Gynecology, Michigan Statc University, Eust Latzsing The time between administration of human chorionic gonadotropin (hCG) and follicle aspiration was analyzed for alterations in L3H]uridine incorporation [as a n indicator of relative synthesis of ribonucleic acid (RNA)] of oocytes from squirrel monkeys and humans. There was a significant decline in both the uptake and incorporation of [3H]uridine in squirrel monkey oocytes by 36 h r following hCG administration t o the animals, as compared with 16 hr after hCG. Similarly, RNA synthesis diminished in oocytes collected 35 hr after hCG in humans, as compared with 12 hr after hCG. This reduction in RNA synthesis of maturing oocytes is similar to that of other mammalian species. This provides evidence for an increased interval between hCG administration and follicle aspiration in order to recover mature oocytes for in vitro fertilization studies. Key words: squirrel monkey, Saimiri sciureus, human, oocytes, human chorionic gonadotropin, ribonucleic acid INTRODUCTION Measurements of the synthesis of macromolecules [e.g., ribonucleic acid (RNA)] have provided indications of the overall metabolic competency of the mammalian oocyte. Because of the scarcity of material available from humans or nonhuman primates, most biochemical studies have been carried out on ova from murine species. However, through the use of laparoscopic procedures for oocyte collection, we can now make such essential biochemical determinations on individual oocytes from primates [Chan et al, 19821. We therefore compared alterations in the incorporation of [3H]uridine between oocytes collected laparoscopically from Saimiri and human females. We also evaluated the effect of the length of the interval between the administration of human chorionic gonadotropin (hCG) to induce ovulation and follicle aspiration on relative RNA synthesis. As RNA synthesis appears to decline Received May 14,1983;accepted June 26,1983. Address reprint requests to Dr. R.J. Hutz, Endocrine Research Unit, Michigan State University, East Lansing, MI 48824. 0 1983 Alan R. Liss, Inc. 268 Hutz, Holzman, and Dukelow with follicle development prior to maturation of the mouse oocyte (i.e., resumption of meiotic progression as a necessary prerequisite of fertilization) [Baker et al, 1969; Wolgemuth-Jarashow and Jagiello, 19791, we wished to determine if these same changes occurred in squirrel monkeys and humans. MATERIALS AND METHODS Subjects Twenty adult nulliparous female squirrel monkeys of Bolivian origin (Primate Imports, Port Washington, NY) and weighing between 600 and 800 g were housed five to six per cage and fed under conditions described previously [Kuehl and Dukelow, 19791. Animals were used during the breeding season and received an ovulatory regimen of four days of 1 mg of follicle stimulating hormone (FSH) IM daily (Burns-Biotec Laboratories, Inc., Omaha, NE). This reflects the hormonal regimen designed for induction of single and double ovulations in squirrel monkeys [Dukelow, 19701. On the final day of FSH treatment, 250 IU hCG was administered IM either 16 or 36 h r prior to laparoscopy for follicular aspiration [Dukelow, 19791 to recover immature or mature oocytes, respectively. Squirrel monkey oocytes were recovered at laparoscopy and mechanically denuded of adhering cumulus cells with glass pipettes. In the human studies, two patients were administered 5000 IU hCG IM on day 12 of the menstrual cycle to induce ovulation. Laparoscopic procedures and oocyte collection were routine [Jones et al, 19821. RNA Synthesis Immature oocytes or mature ova from squirrel monkeys (which were viable as assessed by vital dye staining [Chan et al, 19821 were incubated in 0.25 ml of 5.6 pM 5-[3H]uridine (SA 18 Ci/mmole) added to the culture medium (TC-199, GIBCO, Grand Island, NY; modified with 1 mM pyruvate, 100 pg/ml gentamycin and 20% fetal calf serum). The concentration of [3H]uridine used produced maximum incorporation by oocytes in preliminary trials. Incubations were for 3 hr in 5% COz in air. Oocytes were washed ten times in medium containing unlabeled uridine in 1000fold excess, solubilized, and analyzed as previously described [Fishel and Surani, 19781. Autoradiography Human oocytes that were viable according to vital dyes [Chan et al, 19821 were incubated for 3 hr in modified TC-199 supplemented with 5.6 pM 5-[3H]uridine(SA 18 Ci/mmole) and treated as described above. Following washing, ova were fixed 24 hr in Bouin’s fluid and processed for autoradiography [Weitlauf and Greenwald, 19711. Ova were embedded in paraffin. Autoradiographs were prepared as described previously [Chan et al, 19821. Statistical Analyses Since scintillation counts normally follow a Poisson distribution [Steel and Torrie, 19801 such data were transformed(&) prior to further analyses. Comparisons were analyzed by Student’s t test. P < 0.05 was considered significant. RESULTS The incorporation and uptake of [3H]uridine were both significantly reduced in oocytes recovered from squirrel monkeys 36 hr after hCG administration (which had extruded the first polar body) as compared with oocytes collected 16 hr after hCG (with intact germinal vesicles) (Table I). RNA Synthesis in Primate Ooeytes 269 TABLE I. ["HIUridine Incorporation With Respect to the Time of Oocyte Collection in the Squirrel Monkey Treatment Incorporationa 16 hr after hCG 36 hr after hCG 3.81 f 0.54 (8p 1.07 f 0.14 (4)* Uptakea 68.41 f 8.93 (8) 3.28 f 1.00 (4)* aFemtomoles [3H]uridine/oocyte/3 hr i SEM. bNumber of trials in parentheses; one to five oocytes per trial. *Significantly different from respective group a t 16 hr (P < 0.05). TABLE 11. ["HIUridine Incorporation With Respect to the Time of Oocvte Collection in Humans Treatment Grains/1,000 pm2 ooDlasma 12 hr after hCG (patient 002) 35 hr after hCG (patient 005) 21 -b aOva were incubated for 3 hr in 5.6 pM ['Hluridine. bNot statistically different from background levels. Autoradiographic determinations of counts of silver grains in human oocytes showed a reduced incorporation of 13H]uridine by 35 h r after hCG administration compared to 12 h r after hCG (Table 11).The oocytes collected from the two human patients were aspirated from large follicles approximately 20 mm in diameter. The oocyte recovered 12 h r after hCG retained a germinal vesicle and a prominent nucleolus. This is indicative of a n immature state. In contrast, the oocyte recovered 35 hr after hCG had undergone dissolution of the germinal vesicle and was therefore presumed to have resumed its meiotic progression. DISCUSSION This is the first report of discernible alterations i n relative RNA synthesis with oocyte maturation in primates. With only one human oocyte per treatment, statistical evaluations were not done. However, there is a n apparent decrease in incorporation of [3HJuridine at 35 hr after hCG. This timing is coincident with that of oocyte collection for in vitro fertilization in humans [Trounson et al, 19811. The decrease in t3H]uridine incorporation by oocytes 36 h r after hCG in the squirrel monkey is consistent with that seen with intrafollicular maturation in oocytes of other mammalian species [Baker et al, 1969; Wassarman and Letourneau, 19761. The diminished synthesis of RNA in squirrel monkey and human oocytes indicates a more mature state for the oocyte (of critical importance for in vitro fertilization). From a biochemical viewpoint, a n increased time interval between hCG and oocyte collection is preferred if long culture periods in vitro are ineffective in oocyte maturation. The present study demonstrated alterations in biochemical events during oocyte maturation of both squirrel monkeys and humans similar to those reported for other mammalian species. CONCLUSIONS 1. Relative RNA synthesis in oocytes decreased with increased time after hCG administration to both squirrel monkeys and humans. 2. The biochemical alterations discovered in primate oocytes are similar to those found in other mammalian species. 270 Hutz, Holzman, and Dukelow ACKNOWLEDGMENTS The authors wish to thank Mrs. L.M. Cleeves for her assistance with the manuscript and Dr. P.J. Chan for providing the squirrel monkey ova. The study on human oocytes was approved by the Human Subjects Committees of Michigan State University, East Lansing, and Edward W. Sparrow Hospital, Lansing, Michigan. This research was supported, in part, by grants from the National Institutes of Health and the Health Science Research Foundation. REFERENCES Baker, T.G.; Beaumont, E M . ; Franchi, L.L. The uptake of tritiated uridine and phenylalanine by the ovaries of rats and monkeys. JOURNAL OF CELL SCIENCE 4:655-675, 1969. Chan, P.J.; Hutz, R.J.; Dukelow, W.R. Nonhumsn primate in vitro fertilization: Seasonality, cumulus cells, cyclic nucleotides, ribonucleic acid, and viability assays. FERTILITY AND STERILITY 381609-615, 1982. Dukelow, W.R. Induction and timing of single and multiple ovulations in the squirrel monkey (Saimiri sciureus). JOURNAL OF REPRODUCTION AND FERTILITY 22~303-309, 1970. Dukelow, W.R. Human chorionic gonadotropin: Induction of ovulation in the squirrel monkey. SCIENCE 206234-235,1979. Fishel, S.B.; Surani, M.A.H. Changes in responsiveness of preimplantation mouse embryos to serum. JOURNAL OF EMBRYOLOGY AND EXPERIMENTAL MORPHOLOGY 45295-301, 1978. Jones, H.W.; Jones, G.S.; Andrews, M.C.; Acosta, A,; Bundren, C.; Garcia, J.;Sandow, B.; Veek, L.; Wikes, C.; Witmeyer, J.; Wortham, J.E.; Wright, G. The program for in vitro fertilization at Norfolk. FERTILITY AND STERILITY 38:14-21.1982. Kuehl, T.J.; Dukelow, W.R. Maturation and in vitro fertilization of follicular oocytes of the squirrel monkey (Saimiri sciureusi. BIOLOGY OF REPRODUCTION 21:545-556, 1979. Steel, R.G.D.; Torrie, J.H. PRINCIPLES AND PROCEDURES OF STATISTICS. A BIOMETRICAL RESEARCH. New York, McGraw-Hill, 1980. Trounson, A.O.; Leeton, J.F.; Wood, C.; Webb, J.; Wood, J. Pregnancies in humans by fertilization in vitro and embryo transfer in the controlled ovulatory SCIENCE . cycle. . 212681-682,1981. Wasserman. P.M.: Letourneau, G.E. RNA synthesis 'in fully-grown mouse oocytes. NATURE 26133-74,1976. Weitlauf, H.M.; Greenwald, G.S. Preparation of preimplantation embryos for autoradiography, pp 278-283 in METHODS IN MAMMALIAN EMBRYOLOGY. J.C. Daniel, ed. San Francisco, W.H. Freeman, 1971. Wolgemuth-Jarashow, D.J.; Jagiello, G.M. RNA synthesis during in vitro maturation of mammalian oocytes, pp 379-383 in OVARIAN FOLLICULAR DEVELOPMENT AND FUNCTION. A.R. Midgley and W.A. Sadler, eds. New York, Raven Press, 1979.