Ooplasmic transfusion: prophase germinal vesicle oocytes made developmentally competent by microinjection of metaphase II egg cytoplasm*†

Assisted reproductive technology FERTILITY AND STERILITY Copyright

©

Vol. 53, No.6, ,June 1990

Printed on m·id-free paper in U.S.A.

1990 The American Fertility Society

Ooplasmic transfusion: prophase germinal vesicle oocytes made developmentally competent by microinjection of metaphase II egg cytoplasm*t

Jill T. Flood, M.D.:j: Claudio F. Chillik, M.D.§ Jan F. H. M. van Uem, M.D.[[

Akira Iritani, Ph.D. 'II Gary D. Hodgen, Ph.D.:j:#

The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, Virginia; Centro de Estudios Ginecologicos y Reproducion, Buenos Aires, Argentina; Erlangen, Den, West Germany; and College of Agriculture, Kyoto University, Kyoto, Japan

Approximately one fourth of all human oocytes collected for in vitro fertilization are of immature origin. Even when these oocytes undergo nuclear maturation, fertilization, and cleavage in vitro, transfer of such embryos rarely results in pregnancy reaching delivery. We hypothesized that human embryos derived from prophase I oocytes were developmentally incompetent because they lacked a factor(s) found in in vivo matured oocytes. Using micromanipulation techniques in monkeys, we removed ooplasm from metaphase II oocytes and injected it into prophase I oocytes. After nuclear maturation, oocytes were transferred to the fallopian tube for fertilization. After ooplasmic transfusion, prophase I oocytes resulted in a delivery rate of 13%. When metaphase II ooplasm was heated or exposed to ribonuclease A before microinjection into prophase I oocytes, it lost effectiveness in conferring developmental competence. Fertil Steril53:1049, 1990

Gonadotropic stimulation for multiple follicular development in in vitro fertilization and embryo transfer (IVF-ET) or gamete intrafallopian transReceived May 23, 1989; revised and accepted February 17, 1990. *Presented in part at the 35th Annual Meeting of the Society for Gynecologic Investigation, Baltimore, Maryland, March 17 to 20, 1988. t Supported in part by a Barlex Scholar Award, New York, New York. :j: Department of Obstetrics and Gynecology, The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School. § Centros de Estudios Ginecologicos y Reproducion. II Erlangen, Den, West Germany. 'II Department of Animal Sciences, College of Agriculture, Kyoto University. # Reprint requests: Gary D. Hodgen, Ph.D., Professor and Scientific Director, The Jones Institute for Reproductive Medicine, and Director, Contraceptive Research and Development (CONRAD) Program, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, 855 West Brambleton Avenue, Suite B, Norfolk, Virginia 23510. Vol. 53, No.6, June 1990

fer programs typically leads to the development of a quasi -synchronous cohort of oocytes in various maturational stages. Aspirated oocytes range from postmature metaphase II to mature metaphase II and metaphase I, which often yield viable pregnancies, and immature prophase I oocytes with a germinal vesicle. Condition of the cumulus cell mass is frequently indicative of oocyte status. 1 In the Norfolk IVF-ET program, immature oocytes make up approximately one fourth of all human oocytes collected (Table 1). Despite subsequent nuclear maturation achieved in vitro, in fertilization, and in cleavage of some prophase I oocytes, these embryos rarely contribute to viable pregnancy. Thus, efforts to mature germinal vesicle oocytes in vitro have not adequately improved their developmental potential. Several reports of cytoplasmic factors having either unspecified functions or cell cycle regulatory roles have been reported. 2- 8 When egg cytoplasm from cells exhibiting a specific function are miFlood et al.

Ooplasm transfer to prophase oocytes

1049

Table 1

Outcome of8,816 Oocytes From 1,397 Treatment Cycles• Collected for IVF, January 1985 to December 1987b

No. of oocytes

Oocyte classification

Metaphase II Preovulatory unclassified Metaphase I Prophase I Fractured zona Degenerate

2,980 (33.8) 604 (6.9) 1,960 (22.2) 2,347 (26.6) 531 (6.0)} 394 (4.5)

Nuclear maturation in vitroc

Fertilization rate/ insemination d

%

%

100 84.7 Research use only

92.3} 91.0 86.4 51.5

Pregnancies/ transfer cycle e

3~5/1,359

(28.3)

1/38 (2.6)

Pregnancies/''pure'' transfer cycle t

106/388 (27) 12/68 (19) 20/92 (22) 1/38 (2.6)

• Each treatment cycle means the patient reached egg collection. Includes all patient and donor recipient cycles, except those with severe male factor. b Values in parentheses are percents. c Nuclear maturation in vitro of metaphase I oocytes is statistically greater than that for prophase I oocytes; P < 0.001 (x 2 ). d Fertilization rate per oocyte inseminated is not statistically different between metaphase II and preovulatory unclassified oocytes at retrieval, but is significantly greater for metaphase ll and preovulatory unclassified oocytes than for metaphase I oocytes and prophase I oocytes. In addition, the fertilization rate per oocyte inseminated for metaphase I oocytes is statistically greater than that for prophase I oocytes; P < 0.001 (x 2 ).

• Pregnancies established per embryo transfer cycle of metaphase II, preovulatory unclassified, and metaphase I oocytes at the time of oocyte collection is statistically greater than that for oocytes collected in the prophase I stage; P < 0.001 (x 2 ). t Pregnancies established per embryo transfer cycle of embryos originating from only metaphase II oocytes or from preovulatory unclassified oocytes or metaphase I oocytes is not statistically different, but is statistically greater for each of those groups than for oocytes collected in the prophase I stage; P < 0.001 (x 2 ). Modified from: Veeck LL: Oocyte assessment and biological performance. In In Vitro Fertilization and Other Assisted Reproduction, Edited by HW Jones, Jr., C Schrader. Annals of the New York Academy of Sciences, 1988, p 259

croinjected into cells lacking that function, the injected cells often exhibit the transferred function, whether it is inhibitory or stimulatory. 2·4·6-8 The entity(ies) transferred may influence genetic, cytoplasmic, or membranous functions, imparting maturational or developmental properties, or inhibit the same. Here, we hypothesized that the cytoplasmic compartment of in vivo matured metaphase II oocytes contains a presynthesized factor(s) required for early cleavage events or eventual coactivation with the paternal genome, 9 - 11 thereby allowing continued embryonic developmental competence. That developmental failure of primate embryos derived from immature oocytes (prophase I) "matured" in vitro may be because of the deficiency of this requisite factor(s)-thus deserving some scrutiny. The present study presents our initial findings using a micromanipulation technique we call ooplasmic transfusion, whereby a small amount of ooplasm is borrowed from an egg that achieved maturity in vivo metaphase II and is introduced into an immature oocytes prophase I subsequently matured in vitro and transferred to a female to determine oocyte developmental competence.

cles (27 to 32 days) and no previous exposure to exogenous gonadotropins were used. Primate husbandry techniques have been previously described.12

MATERIALS AND METHODS

Primates

Thirty-six adult female cynomolgus monkeys (Macaca fascicularis) having regular menstrual cy1050

Flood et al.

Ooplasm transfer to prophase oocytes

Follicular Stimulation and Oocyte Culture

Gonadotropic stimulation of follicular development was performed using human menopausal gonadotropin (Pergonal; Serono Laboratories, Inc., Randolph, MA) as previously described. 13 Then 34 to 36 hours after human chorionic gonadotropin (hCG) administration, follicles were aspirated into Biggers, Whitten, and Whittingham (BWW) media during laparotomy or laparoscopy. 14 Media was made in our laboratory and buffered to pH 7.4. No monkey received human gonadotropin stimulation in more than one menstrual cycle. Oocytes were identified and evaluated according to cumulus cell mass (magnified 400X) and after treatment with 0.1% hyaluronidase, classified by nuclear status (Sigma Chemical Co., St. Louis, MO) according to the criteria of Veeck. 1 Oocytes were cultured in BWW supplemented with 3% bovine serum albumin at 37•c in an atmosphere of humidified 5% C0 2 and air, as previously described.13·14 We obtained 6.3 ± 1.6 usable oocytes per female; slightly more were prophase I than metaphase II stage. All metaphase I oocytes were used in other experiments; if no polar body could be found, we removed the oocyte from the study. Fertility and Sterility

Microinjection Equipment

Pipets of similar tip volume (about 15 pL) were selected by prior comparisons using a radioactive solution. Tip capacities varying >50% from the mean were discarded. All injection procedures were carried out on aNikon diaphot inverted microscope (Image Systems, Inc., Columbia, MD) equipped with Narishige micromanipulators (Narishige Co., Ltd., Tokyo, Japan). Injection and egg holding pipets were prepared using a Narishige pp-93 micropipet puller and MF-79 micropipet forge (Narishige). The inside diameter of the microinjection pipet tip was 8 to 10 JLID. The injection pipet tip was broken to yield a sharp tip by placing the tip into the opening of the holding pipet and rapidly rotating the micromanipulation. The tip angle was about 45oC and used unpolished. All microinjection procedures were performed with 200 to 300 JLL ofBWW media (37°C) under heavy mineral oil to minimize evaporation and rapid pH change. Study Design: Experiment I

After nuclear classification, 10 to 20 pL of ooplasm was aspirated from metaphase II oocytes (Fig. 1). This ooplasm was then injected into one or two prophase I oocytes, from the same monkey whenever possible. Care was taken to minimize leakage of cytoplasm. After ooplasmic transfusion, oocytes were incubated in culture media until they had extruded the first polar body (up to 24 hours). Metaphase II oocytes were cultured for an additional 6 hours to allow "healing" of those with depleted ooplasm. One to three oocytes were transferred by laparotomy to the fallopian tube 15 of castrate, steroid-replaced16 mated recipients. Oocytes of only one nuclear classification at aspiration and of one treatment or control group were transferred per recipient. There was not an imbalance of multiple oocyte transfers. Metaphase II stage oocytes in triple transfer were done only once more than prophase I stage oocytes. There were four control groups: metaphase II and prophase I oocytes undergoing no manipulation, but with environmental exposure and culture conditions similar to manipulated oocytes; and metaphase II and prophase I oocytes undergoing sham puncture without depletion or injection of ooplasm.

Figure 1 Metaphase II oocyte (top) immediately before penetration by aspiration micropipet, and (bottom) with micropipet centrally within during aspiration of ooplasm (original magnification was X400).

minutes before transfusion into prophase I oocytes. A small drop of oil was drawn into the tip of the micropipet after ooplasm aspiration to prevent evaporation during heating. In a second group of oocytes, we drew ribonuclease A (0.5% RNase in BWW medium; Sigma Chemical Co.) into the aspiration pipet to a level above that usually used for aspiration of ooplasm removal. The RN ase was then ejected from the pipet and the pipet used immediately to aspirate metaphase II ooplasm that was incubated in the RNase coated pipet for 20 minutes at 37oC before ooplasmic transfusion into prophase I oocytes. Oocytes in experiment II were transferred as described for experiment I. Assays

Serum was assayed for monkey chorionic gonadotropin 14 days after tubal transfer of oocytes as previously described. 16

Study Design: Experiment II

Statistical Analysis

As above, we aspirated metaphase II ooplasm into micropipets and heated this to 60oC for 20

Data were compared statistically using x2 analysis, matching both unmanipulated and sham-rna-

Vol. 53, No.6, June 1990

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Ooplasm transfer to prophase oocytes

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Table 2

Ooplasmic Transfusion of Metaphase II Cytoplasm Into Germinal Vesicle Containing Oocytes at Prophase" Metaphase II oocytes

Prophase oocytes Ooplasm transferred (A) Total number of

42

oocytes (B) Oocytes transferred at metaphase II (C) Pregnancies detected by monkey chorionic gonadotropin assay (D) Live born infants

23 (A) (55) 4 (A) (10) (B) (17) 3 (A) (7) (B) (13) (C) (75)

Sham punctured

Nonsurgical controls

Ooplasm aspirated

43

37

22

18

24

26 (60) 0

30 (80) 0

22

18

24

0

0

4 (18) (18) 4 (18) (18) (100)

Sham punctured

Nonsurgical controls

3 (17) (17) 3 (17) (17) (100)

6 (25) (25) 4 (17) (17) (67)

"Numbers in parentheses are percents.

nipulated controls to both metaphase II depleted and prophase I injected groups. RESULTS

Experiment I

Table 2 shows the results of ooplasmic transfusion from metaphase II into prophase I oocytes. Forty-two prophase I oocytes received metaphase II ooplasm. We estimated that 63% survived surgical trauma. Of those ooplasmic transfused prophase I oocytes transferred, four pregnancies (17%) were detected by monkey chorionic gonadotropin. Three of these pregnancies resulted in live single births (live birth/transferred oocyte, 13%). There were no pregnancies from 43 sham punctured or 37 nonsurgical control prophase I oocytes. The rate of nuclear maturation in vitro of punctured prophase I oocytes (transfused, 55%; sham punctured, 60%) was significantly decreased compared with nonsurgical controls (80%; P < 0.05). Among prophase I ooplasmic transfused eggs, the pregnancy rate per transfer cycle was 26%. There were four pregnancies (18%) detected by monkey chorionic gonadotropin from 22 metaphase II oocytes replaced after partial depletion of ooplasm, which was not statistically different from the pregnancy rate of sham-punctured metaphase II oocytes (17%), nonsurgical metaphase II controls (25% ), or importantly, prophase I oocytes receiving ooplasmic transfusion (17%). The live birth rate per oocyte transferred was not statistically different between any groups. The pregnancy rate per transfer cycle for metaphase II oocytes was31%. 1052

Flood et al.

Ooplasm transfer to prophase oocytes

Experiment II

There were no pregnancies detected by monkey chorionic gonadotropin from 13 prophase I oocytes transferred after heat exposed metaphase II ooplasmic transfusion or from 14 prophase I oocytes transferred after RNase exposed ooplasm transfusion (Table 3). DISCUSSION

In the Norfolk IVF program, approximately one fourth of all oocytes collected are prophase I. Eighty-five percent of these oocytes undergo germinal vesicle breakdown with extrusion of the first polar body during extended culture before insemination. Fifty-one percent achieve fertilization; many cleave at least once before uterine transfer. Table 3 Ooplasmic Transfusion of Heat or mRNase Exposed Metaphase II Cytoplasm Into Germinal Vesicle Containing Prophase Oocytes

Heat exposed metaphase II ooplasm transferred (A) Total number of

prophase I oocytes transfused (B) Oocytes transferred at metaphase II (C) Pregnancies detected by monkey chorionic gonadotropin assay

20

13 (A) (65)"

0

mRNase exposed metaphase II ooplasm transferred 22

14 (63)"

0

• Value is a percent.

Fertility and Sterility

In spite of the occurrence of nuclear maturation in vitro and early cleavage events, the rate of pregnancy from oocytes of prophase I origin at harvest is minimal (2.6%) when compared with pregnancies from metaphase I or II oocytes undergoing germinal vesicle breakdown in vivo, before oocyte collection (25.2%). Many oocyte/embryo culture modifications have been tried to improve in vitro oocyte maturation; however, all have relied on nuclear events or rate of fertilization, without achieving evidence of improved developmental competence. Cytoplasmic control of nuclear activities during the cell cycle has been investigated by nuclear transplantation and cell fusion, 3 as well as cytoplasmic transfer. 2•4·6-8 Animal data has shown that many events in the cell cycle are related to cytoplasmic regulatory factors, including germinal vesicle breakdown,2•7•8 chromosomal decondensation,5 and metaphase arrest. 4 •6 These cytoplasmic processes are specific to certain phases of the cell cycle and do not seem to be species-specific. Rather, they may be highly conserved throughout evolution. 5·7•8 •17 In our experiments, ooplasmic transfusion from in vivo nuclear matured oocytes into prophase I oocytes did convey developmental competence to some prophase I oocytes. This observation strongly suggests that there is a cytoplasmic factor(s) essential for continued developmental competence that is lacking in oocytes containing a germinal vesicle at harvest. Preliminary data from experiment II further indicates that this cytoplasmic factor may be heat labile, such as specialized proteins or messenger ribonucleic acid, that directs subsequent cell cycle events. Speculation includes the possibility that post-translational protein modification may be interrupted. Although specific evidence is lacking, we wonder if premature exposure of follicles and oocytes to hCG may preclude appropriate production of the cytoplasmic factor(s) requisite to developmental competence, despite apparent nuclear maturation in vitro. Although no pregnancies occurred from oocytes receiving heat or messenger (m)RNase exposed cytoplasmic transfusions, the numbers in these groups are too small to reach statistical significance. It is possible that injection of small amounts of mRNase into the cytoplasm or heated cytoplasm may have had a toxic effect or digestion action on these oocytes. The rate of extrusion of the first polar body among prophase I punctured versus nonsurgical Vol. 53, No.6, June 1990

prophase I control oocytes was statistically different (P < 0.05). We interpret this difference to indicate some mechanical trauma resulted from the insertion of the micropipet. Although care was taken to avoid the polar body or germinal vesicle during oocyte aspiration or transfusion, respectively, some damage was unavoidable. We cannot rule out parthenogenic activation from this model, except that the infants born were apparently normal males and females. The clinical implications of this preliminary work include the potential that prophase I oocytes harvested during IVF may be made developmentally competent by ooplasmic transfusion from metaphase oocytes, although not severely compromising the developmental potential of ooplasm depleted metaphase II oocytes. However, if such clinical experiments are undertaken, perhaps metaphase II oocytes presenting with either fractured zonae or postmature status are optimal candidates for ooplasmic aspiration. In basic research, we need to isolate and characterize the requisite factor(s) present in the ooplasm of metaphase eggs. Finally, the limited and preliminary nature of these observations must be acknowledged, while we remain interested in pursuing the potential of ooplasmic transfusion.

Acknowledgments. We acknowledge Lucinda Veeck, M.L.T., and Ms. Debbie Jones for their help in computation of IVF data; Susan Lanzendorf, Ph.D., Robert Williams, Ph.D., Lynn Danforth, B.S., and Janice Hammond, B.S., for their many technical contributions; as well as Ms. Martha Forrester for preparation of graphic materials, and Ms. Dara Willett Leary for her editorial contribution and manuscript preparation.

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