Validation of the hemizona assay in a monkey model: influence of oocyte maturational stages*

Transcription

1 FERTILITY AND STERILITY Copyright e 1989 The American Fertility Society Printed in U.S.A. Validation of the hemizona assay in a monkey model: influence of oocyte maturational stages* Sergio Oehninger, M.D.t:j: RichardT. Scott, M.D.t Charles C. Coddington, M.D. Daniel R. Franken, Ph.D. II Anibal A. Acosta, M.D.t Gary D. Hodgen, Ph.D. t The Howard and Georgeanna Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Medical College of Hampton Roads, Norfolk, Virginia; Portsmouth Naval Hospital, Portsmouth, Virginia; and University of the Orange Free State, Bloemfontein, South Africa The hemizona assay (HZA) was used in the monkey model to investigate sperm binding to the zona pellucida and to evaluate binding patterns according to various stages of oocyte development. Adult cynomolgus monkeys were superovulated with human menopausal gonadotropin. The oocytes were retrieved by laparoscopy, stored in salt solution, then cut into hemizonae by micromanipulation. Metaphase II oocytes showed significantly tighter binding than prophase I oocytes (P < 0.001). Metaphase I oocytes showed intermediate binding, significantly different from the other groups (P < 0.01). It is concluded that: (1) This study demonstrates the feasibility of HZA as a test to evaluate sperm/zona interactions in the monkey, using starldards reported for human studies. (2) Oocyte meiotic competence seems to be accompanied by an increase in zona-binding properties. Fertil Steril51:881, 1989 The hemizona assay (HZA) has been introduced as a new diagnostic test for the binding of human spermatozoa to human zonae pellucidae to predict fertilization potential. 1 In the HZA, the two matched hemizonae created by micro bisection provide two surfaces that are functionally equal, thus allowing a controlled comparison of sperm-zona interactions and binding. Previous studies of human oocytes examined the precision of zona cutting, the feasibility of sperm binding to the hemizonae, and the kinetics of tight Received September 19, 1988; revised and accepted December 20, * The opinions expressed herein are those of the authors and do not represent those of the United States Department of the Nayy or the United States Department of Defense. t The Howard and Georgeanna Jones Institute for Reproductive Medicine, Eastern Virginia Medical School. :1: Reprint requests: Sergio Oehninger, M.D., Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, 825 Fairfax Avenue, Norfolk, Virginia Portsmouth Naval Hospital. II Department of Obstetrics and Gynecology, University of the Orange Free State. sperm binding, showing maximal binding after 4 to 5 hours in coincubation. 1 2 In addition, frozenthawed oocytes obtained from surgically removed ovaries showed binding properties equal to unfertilized oocytes discarded from our in vitro fertilization (IVF) program. 1 Furthermore, normal spermatozoa showed equivalent binding to dimethylsulphoxide- (DMSO; 2.0 M solution) stored and salt- (magnesium chloride, MgC12 ; 1.5 M solution) stored human hemizonae. 2 This salt solution offers a simpler way of storing human zonae pellucidae while maintaining functional (binding) properties. All human studies performed so far have used oocytes arrested at the prophase I stage. Prospective blinded studies using such immature oocytes showed that the HZA is a valuable tool for evaluating dysfunctional sperm-zona binding, with good predictive value for IVF outcome. 3 4 Mimicry of the human ovarian/menstrual cycle by nonhuman primates (Macaques) has provided an adequate model for ovarian response to gonadotropin stimulation and for IVF-embryo transfer. 5-7 The monkey model allows researchers the freedom Oehninger et al. HZA, monkey, oocyte maturation 881

2 to pursue aggressive protocols without the ethical constraints that could limit similar clinical studies in humans (i.e., experimental studies performed on preovulatory oocytes from women undergoingivf). The present study was designed to answer the following questions: (1) Can sperm binding to the zona pellucida be evaluated by the HZA in our primate model (cynomolgus monkey)? 6 (2) If so, does the binding pattern differ at various stages of oocyte development (immature versus preovulatory oocytes)? MATERIALS AND METHODS Primate Husbandry Normal adult female and male cynomolgus monkeys (Macaca fascicularis) weighing 3 to 6 kg were caged individually in a controlled environment with the temperature at 75 to so c, humidity 60% minimum, and a 12/12-hour light/dark cycle. 8 The monkeys were given water ad libidum, fed commercial monkey chow twice a day, and maintained by husbandry practices previously described. Females had a history of regular menstrual cycles, and daily vaginal swabs were used to detect the onset and duration of menses. Treatment Protocol Three females were given human menopausal gonadotropin (Serono Laboratories, Randolph, MA) daily, starting on day 3 of their menstrual cycles, at a dose of 37.5 IU /day intramuscularly (IM), for a period of 5 to 6 days. When serum estradiol (E2) levels reached 600 to 1,000 pg/ml, with a shift in biologic cervical mucus and vaginal maturation index, 9 stimulation was stopped. Human chorionic gonadotropin, 1,000 IU, was administered IM 32 to 36 hours thereafter (coasting interval). Thirty-six hours later, laparoscopic oocyte retrieval was performed under ketamine anesthesia. Follicular Aspiration Follicles were aspirated into Dulbecco's phosphate-buffered saline (PBS) using a 10-cc syringe incubated at 37 c. On collection, the follicular fluid was immediately transferred to the culturing laboratory, where examination of the fluid in Falcon no petri dishes (Falcon Plastics, Oxnard, NJ) was undertaken to locate and identify the oocytes, using a dissecting stereomicroscope (Nikon SMZ-10) and an inverted high-resolution phasecontrast microscope (Nikon Diaphot, Garden City, NJ). Oocyte classification was performed according to the criteria of Veeck. 10 Oocyte Storage After determination of the oocyte maturational stage, oocytes were stored at room temperature in capped microcentrifuge tubes containing 0.5 ml of 1.5 M MgCl2, 0.1% polyvinylpyrrolidone, and 40 mm of HEPES buffer. Tubes were kept at room temperature until ready for micromanipulation. Experiments were carried out over a period of 5 to 21 days. Cutting Oocytes into Hemizonae by Micromanipulation Oocyte microbisection was performed under an inverted phase-contrast microscope equipped with a complete micromanipulation system (model MO 102; Narishige, Tokyo, Japan). The micromanipulator was fitted with a Beaver chuck handle (no. 1312, Katena Products, Denville, NJ) with a Beaver micro-sharp blade (no. 7530) attached. A 100 X 20 mm plastic petri dish (Falcon no ) served as the cutting chamber. The dish was filled with 3 to 4 mm of Ham's F-10 with 3.0 mg/ml of bovine serum albumin (BSA). The blade was positioned exactly parallel to the dish. With micro manipulators, the blade was centered in the field of view and slowly lowered to score a groove on the bottom surface of the dish. Approximately 0.5 ml of medium was added to each vial containing the oocytes. Vials were recapped and inverted 15 to 20 times. The contents of the vials were emptied into a 35 X 10 mm plastic petri dish. The oocytes were recovered and rinsed in a drop of medium. Each oocyte was transferred to the working area of the dish by means of a finely drawn glass pipette. The oocyte was positioned on the groove and the blade was centered over the oocyte. With 200X magnification, the blade was slowly lowered, initiating a midline cut into the zona. The blade was continually lowered until two cleanly cut hemizonae were produced. Dense ooplasm was dislodged by vigorous pipetting with a small-bore drawn glass pipette. Each hemizona pair was placed in a drop of medium under heavy white mineral oil (Sigma Chemical Co., St. Louis, MO) in a 35 X 10 mm petri dish and kept at room temperature until ready for use Oehninger et al. HZA, monkey, oocyte maturation Fertility and Sterility

3 Sperm Preparation Semen samples were obtained from a cynomolgus primate via electroejaculation with a rectal probe under ketamine anesthesia. Semen was collected into a 50 ml test tube containing PBS with 3.0 mg/ml of BSA. The average sperm concentration and motility in the specimen used in these experiments were 168 X 10 6 /ml and 80%, respectively. Twenty minutes after collection, a small aliquot of semen was observed by light microscopy to determine semen quality. An aliquot of semen (0.3 ml) was diluted with 0.5 ml of Ham's F-10 with 3.0 mg/ml of BSA and washed by centrifugation (8 minutes at 400 X g). The supernatant was removed and discarded. Pelleted sperm were washed a second time (5 minutes at 400 X g). The second pellet was overlaid with 0.5 ml of Ham's F-10 and incubated at 37"C (5% C0 2 in air) to effect a swimup separation. After 1 hour of incubation, the supernatant was recovered, diluted, and used for thehza. Coincubation with Hemizonae Two 100-~l drops of the final dilution (500,000 motile sperm/ml) were placed in a 35 X 10 mm plastic petri dish. The semen drops were kept under heavy white mineral oil (Sigma Chemical Co., St. Louis, MO). Matching hemizonae were placed in semen drops of the same sample and coincubated at 37"C (5% C0 2 in air) for 4 hours. 2-4 Counting of Tightly Bound Sperm After 4 hours of incubation, the zonae were rinsed in a cold medium drop 5 times with a large drawn glass pipette to dislodge loosely attached sperm. The zonae were transferred to a fresh medium drop and positioned with the outer surface upward. At 200X magnification, the number of tightly bound sperm on the outer surface was counted, then discarded. 1-4 Blood Collections and Hormonal Assays Daily serum samples (3 to 5 ml) were obtained in the female monkeys by femoral puncture from day 2 until the day of oocyte retrieval for rapid E 2 radioimmunoassay determination. 8 Statistical Analysis Data were examined by the two-tailed Student's t-test. The level of significance was set at Table 1 HZA (Tight Sperm Binding) in the Cynomolgus Monkey: Results According to Stage of Oocyte Maturation No. of sperm bound/ matched hemizonae Mean±SE Mean value ofthe difference between the two halves Prophase I (n= 8 oocytes, 16 hemizonae) ± ± 3.9 Metaphase I Metaphase II (n =6 (n= 7 oocytes, oocytes, 12 hemizonae) 14 hemizonae) "/ ± 5.4" 68.9± ± ± 0.8' P < 0.01 compared with prophase I and metaphase II oocytes. P < compared with prophase I oocytes. ' No significant differences among the groups. Results are expressed as the mean ± standard error (SE). RESULTS A total of 21 oocytes were obtained after gonadotropin stimulation and retrieval in the three female monkeys. Of these, 8 were classified as immature (prophase I) oocytes, 6 as metaphase I, and 7 as metaphase II, which provided 16, 12, and 14 hemizonae, respectively, to be used as matched pairs in thehza. The mean number of tightly bound sperm was 21.1 ± 3.4 for prophase I, 44.0 ± 5.4 for metaphase I, and 68.9 ± 5.8 for metaphase II oocytes. Mature (metaphase II) oocytes had significantly more tightly bound sperm than the immature (prophase I) oocytes (P < 0.001) (Table 1). Metaphase I oocytes had significantly higher binding than prophase I oocytes, but less than metaphase II oocytes (P < 0.01). The mean value of the difference between the two halves (matching hemizonae) for each maturation stage was 10.1 ± 3.9 for prophase I, 12.3 ± 2.0 for metaphase I, and 14.2 ± 0.8 for metaphase II oocytes. There were no significant differences between these values in comparing immature and mature oocytes, thus showing a small intra-assay variation for all maturational stages. DISCUSSION These results demonstrate the feasibility of the HZA as a test to evaluate tight sperm binding to Oehninger et al. HZA, monkey, oocyte maturation 883

4 the zona pellucida in the cynomolgus monkey, using standards reported for human studies.1-4 Even though further experiments are required for elucidation of binding kinetics, interegg variation and alternative oocyte storage conditions (such as DMSO), these results open a novel avenue for animal gamete research using the HZA for evaluation of sperm-zona interaction and spermatozoa ( dys )function. Mammalian zona pellucida is an extracellular matrix of glycoproteins unique to the oocyte. 11 Specific in vivo functions assigned to the zona include sperm binding, induction of the acrosome reaction, and involvement in the "slow" block to polyspermia Therefore, the zona pellucida is intimately involved with the fertilization process. O'Rand et al.15 have proposed that the specificity at the level of sperm-zona interaction lies in the ability of the sperm to bind and penetrate the zona and that this penetration has two requirements: (1) the high affinity of the zona-binding protein for the sperm receptor and (2) the necessity of having bound zona protein removed or degraded from the sperm surface. The HZA offers the arena in which kinetics of sperm binding, binding patterns of normal and abnormal sperm, percentage of acrosome-reacted sperm bound to the HZA, and zona penetration can be elucidated in the nonhuman and human species. Recent studies16 using immunocytochemical techniques have demonstrated that the formation of the rabbit zona pellucida is initiated just before the differentiation of the granulosa cells. During oogenesis, the size of the oocyte increases dramatically. In the mouse, the presence of the zona pellucida has been observed in oocytes with a diameter of 30 #tm,17 and its production continues in oocytes as large as 70 #tm.18 This growth represents at least a fivefold increase in the surface area of the oocyte. However, the encompassing zona pellucida is a metabolically stable structure with a long halflife.19 Thus, the fenestrated network observed in the zonae of ovulated eggs may indicate flexibility of the earliest laid-down zona proteins that accommodate the tremendous oocyte growth. 20 During oogenesis, meiotic competence (and thus fertilizability) is accompanied by significant changes in metabolic activity and ultrastructure However, it appears that ovulated eggs no longer synthesize zona proteins.17 In our experiments, oocytes arrested at the dietyate stage of the first meiotic prophase (immature) show significantly less tight sperm binding than meiotically competent metaphase II (mature) oo- 884 Oehninger et al. HZA, monkey, oocyte maturation cytes. In our experience with gonadotropin stimulation in the human and in the monkey, antral follicles provide immature and mature oocytes that do not differ in diameter. Chan22 also reported that neither the mean ooplasma radius nor the mean zona thickness differs in a comparison of mature with intermediate and immature human IVF oocytes. Chan showed a positive relationship between oocytes with a thick zona at laparoscopy and the potential for cell division and subsequent embryonic development. If we extrapolate our data from bisected hemizonae to intact oocytes, it may be hypothesized that mature oocytes have a greater ability to bind sperm because of special rearrangements of zona glycoproteins during the late stages of maturation. Further biochemical studies are needed to test this hypothesis, as well as the physiologic mechanism(s) regulating these changes (luteinizing hormone surge?). Recently Familiari et al. 23 reported different sperm-binding capabilities of the zonae pellucidae of human immature and mature oocytes. In accordance with our results, these authors also suggested that the condensation of the outer aspect of the zona, as seen in immature oocytes, causes a disorientation of sperm-binding sites, thus determining less binding. Our previous studies and the present results indicate that the HZA may provide a unique tool to address fundamental questions of the mammalian {primate) fertilization process, with the potential for broader impact on the treatment of human infertility. Acknowledgments. We thank Deborah Johnson, M.A., for technical assistance with the HZA; Jill T. Flood, M.D., Ms. Lynn Sharpe, and Ms. Janice Hammond for surgical assistance, primate husbandry, and hormonal assays; and Charlotte Schrader, Ph.D., for editorial preparation of this manuscript. REFERENCES 1. 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