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Brief report Synthesis of ribonucleic acid in oocytes collected from squirrel monkeys and humans following chorionic gonadotropin administration.

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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.
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PROCEDURES OF STATISTICS. A BIOMETRICAL RESEARCH. New York, McGraw-Hill, 1980.
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Wasserman. P.M.: Letourneau, G.E. RNA
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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.
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acid, chorionic, report, oocytes, following, administration, brief, human, collected, synthesis, monkey, ribonucleic, squirrel, gonadotropin
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