Enhancing in vitro fertilization of mouse oocytes by partial zona pellucida digestion

Journal of Assisted Reproduction and Genetics, Vol. 9, No. 2, 1992

ANIMAL INVESTIGATIONS

Enhancing in Vitro Fertilization of Mouse Oocytes by Partial Zona Pellucida Digestion N. MORDEL, 1'3 S. OHAD, 1 B. ZENTNER, 1 J. G. SCHENKER, 1 J. GORDON, 2 and N. L A U F E R 1

SEM demonstrated that the fine meshlike structure of the outer surface of zona pellucida digested away, leaving a smoother surface. These morphologic changes were not associated with a diminution in sperm binding or penetration. This work demonstrates that partial zona digestion, which causes uniform dissolution of the zona pellucida and reduction of its thickness, is simple and safe. The procedure significantly increased fertilization efficiency at very low sperm concentrations and could, by itself or in conjunction with other methodologies, improve the reproductive capacity of men producing sperm with a reduced penetrating ability.

Submitted: December 12, 1990 Accepted: August 29, 1991

This work was undertaken in order to evaluate the effect of partial zona digestion on fertilization in vitro of mouse oocytes and assess zona surface changes induced by the procedure. Three hundred forty-six oocytes allocated for treatment were exposed to Ham's F-IO medium supplemented with 0.5% Pronase for either 3 min (188 oocytes) or 5 min (158 oocytes); 324 oocytes served as controls. Oocyte losses incurred as a result of the procedure were small (15 oocytes; 4.3%). Control and Pronase-treated oocytes were each divided into four subgroups and inseminatedwith 5 x 105, 5 x 104, 5 x 103, or5 × 102 sperm cells/ml. Fertilization was assessed 8 hr following insemination by the appearance of two pronuclei and development to the two- to four-cell stage the following day. The morphology of the zona pellucida following Pronase treatment was assessed by phase-contrast and scanning electron (SEM) microscopies performed immediately after treatment. Fertilization rate of control oocytes was 80% at a sperm concentration of 5OO,OOO/mland gradually declined to -30% at 500 cells/ml. In contrast, treated oocytes inseminated with 500 sperm cells/ml demonstrated a normal rate of fertilization. At this low sperm concentration the longer Pronase treatment was significantly (P < 0.05) more efficient in enhancing fertilization (69 and 88% for 3 and 5 min of Pronase treatment, respectively). Polyspermic fertilization was not observed in any of the subgroups. Phase-contrast microscopic examination of oocytes at the time of Pronase treatment showed an initial swelling of the zona pellucida for 30--60 sec with a time-dependent increase in its transparency.

KEY WORDS:partial zona pellucidadigestion;in vitro fertilization mouse.

INTRODUCTION Male-factor infertility patients are currently treated by in vitro fertilization (IVF) at an increasing rate because it is assumed that the controlled laboratory conditions offer a better chance for sperm-egg interaction than those provided by intercourse or various forms of insemination. Sperm profiles of infertile males are usually characterized by a low number, abnormal morphology, and reduced motility. In these cases the likelihood that a sufficient number of cells capable of traversing the cumulus oophorus and zona pellucida is reduced, and consequently the male is functionally infertile even if some normal cells are present. Since low sperm motility and a high percentage of abnormal forms have been associated with poor in vitro fertilization rates, routine IVF is effective for only a fraction of oligospermic males (1,2). In an effort to circumvent the barriers to fertilization and assist defective sperm to reach ooplasm,

In Vitro Fertilization Unit, Depaitment of Obstetrics and Gynecology, Hebrew University Hadassah Medical Center, Jerusalem, Israel. 2 Mount Sinai Hospital, New York, New York. 3 To whom correspondence should be addressed at OB/GYN Department, Hebrew University Hadassah Medical Center, Kiryat Hadassah, P.O.B. 12000, IL-91120, Jerusalem, Israel.

1038-0468/92/0400-0128506.50/0 © 1992PlenumPublishingCorporation

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various micromanipulative techniques have been used. Subzonal insertion of sperm involves microinjection of sperm under the zona and then allowing the normal process of fusion to take place (3-6). Zonal drilling (7,8) and partial zona dissection (9) are procedures in which a hole of some kind is created in the zona pellucida to facilitate sperm-egg interaction. These procedures, which eliminate to varying extents the natural process of sperm selection, are aggressive and have so far produced relatively poor results. An alternative and less aggressive approach to enhance fertilization was suggested by Kiessling et al. (10). They have used mild trypsinization to attenuate the zona and improve fertilization of oocytes from three patients with a history of fertilization failure. None of the fertilized oocytes continued to develop after this treatment. This preliminary observation was not investigated further. This work was undertaken in order to evaluate, in the mouse animal model, the efficacy of partial zona digestion in enhancing fertilization in vitro and assess zona surface changes induced by the procedure.

MATERIALS AND METHODS C57B16xBalbc female mice 5-10 weeks of age were superovulated with 12 IU of pregnant mare's serum (Chrono-gest, Intervet, International B.V., Boxmeer-Holland), followed 48 hr later by 12 IU of human chorionic gonadotropin (hCG; Pregnyl, N.V. Organon, Oss, Holland). On the morning following hCG injection animals were sacrificed by cervical dislocation and the cumulus masses were released into Ham's F-10 medium (GIBCO Laboratories, Grand Island, NY). The cumulus cells were removed by gentle mechanical pipetting in Ham's F-10 medium supplemented with hyaluronidase (bovine testes, lyophilized powder, Sigma Chemical Co., St Louis, MO), 2 mg/ml. The cumulus-free oocytes were rinsed several times in fresh medium and moved in batches of about 30 oocytes to 2 ml of medium in organ culture dishes (7029 Falcon, Oxnard, CA). Only oocytes with a polar body and a normal-looking cytoplasm were included. For achieving partial zona pellucida digestion, oocytes allocated for treatment were exposed to Ham's F-10 medium supplemented with 0.5% Pronase (Pronase

Journal of Assisted Reproduction and Genetics, Vol. 9, No. 2, 1992

E, Protease, Type XXV, from Streptomyces griseus, Sigma Chemical Co., St. Louis, MO) for either 3 min (188 oocytes) or 5 min (158 oocytes). Mature fertile males were sacrificed and sperm expressed from sliced epidydimides and vasa defferentia into Ham's F-10 medium supplemented with 5 mg/ml bovine serum albumin (Fraction V, Sigma Chemical Co., St. Louis, MO) for 2 hr. The sperm were not washed prior to insemination. Control and Pronase-treated oocytes were each divided into four subgroups and inseminated at the following sperm concentrations: 5 x 105, 5 x 10 4, 5 X 103, or 5 X 102 sperm cells/ml. Fertilization was assessed 8 hr following insemination by the appearance of two pronuclei and development to the two- to four-cell stage the following day. Scanning Electron Microscopy (SEM) In order to characterize the morphology of the zona pellucida following Pronase treatment, SEM of treated and control oocytes was performed immediately after treatment, and oocytes were fixed in 1% glutaraldehyde for 1 hr and in a 2% solution for another hour. After fixation, oocytes were placed on a coverslip which had been precoated with poly-L-lysine hydrobromide (Sigma Chemical Co.) and refixed with 1% osmium tetroxide in 0.1 M cacodylate buffer for 1 hr (11). Coverslips with the attached oocytes were then dehydrated through a graded series of ethyl alcohol. Specimens underwent a critical-point dehydration with CO2, were coated with gold in an Polaron ES-100 sputter coater, and were examined with a JEM-200CX scanning electron microscope.

RESULTS Fertilization

A total of 346 oocytes underwent partial digestion for either 3 (188) or 5 (158) min. Oocyte losses incurred as a result of the procedure were few (15 oocytes) and due to extrusion of ooplasm through a defect created in the zona by rough mechanical manipulation during the procedure or complete digestion of the zona and oocyte. Fertilization rate of control oocytes was 80% at a sperm concentration of 500,000/ml and gradually declined to - 3 0 % at 500

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cells/ml. In contrast, treated oocytes both at 3 and at 5 min of Pronase exposure inseminated with 500 sperm cells/ml demonstrated a rate of fertilization (69 and 88%, respectively) comparable to that obtained with untreated oocytes at a sperm concentration three orders of magnitude higher (Table I). Polyspermic fertilization was not observed in any of the subgroups. These results suggest that Pronase treatment is capable of enhancing fertilization at very low sperm concentrations.

Morphologic Examination of the Oocytes P h a s e - c o n t r a s t microscopic examination of oocytes at the time of Pronase treatment showed an initial swelling of the zona pellucida for 30--60 sec followed by a slow process of thinning. The thinning was moderate (one-fourth to one-third of the zona width) at 3 min and substantially more pronounced (about one-half) during a 5-min period of pronase digestion. SEM demonstrated that the fine meshlike structure of the outer surface of zona pellucida digested away, leaving a smoother surface (Figs. 1A and B). However, these morphologic changes were not associated with a diminution in sperm binding or penetration (Table I).

DISCUSSION Our work demonstrates that partial zona digestion, which causes uniform dissolution of the zona pellucida and reduction of its thickness, is simple and safe. The data obtained show that the proceTable I. The Effect of Pronase Treatment on Fertilization of Mouse Oocytes Fertilized oocytes Sperrn/ml

Oocyte number

Number (%)

Fertilized oocytes Oocyte number

Number (%)

5 5 5 5

Pronase, 3 min (188 oocytes) x 105 45 37 (82) x 104 49 39 (80) x 103 46 31 (67) x 102 48 33 (69)*

Control (196 oocytes) 51 40 (78)* 48 31 (65) 47 24 (51) 50 18 (36)*

5 5 5 5

Pronase, 5 rain (158 oocytes) x 105 37 29 (78) × 104 41 31 (76) x 103 39 32 (82) × 102 41 36 (88)*

Control (146 oocytes) 38 31 (82)* 33 21 (64) 35 19 (54) 40 10 (25)*

* P < 0.01.

dure did not adversely affect fertilization at normal sperm concentrations used in IVF and significantly increased fertilization efficiency at very low sperm concentrations. These results employing normal sperm and zona thinning are similar to those achieved with normal sperm and zona drilling (7). In the latter a more aggressive methodology creating a hole in the zona pellucida by acid Tyrode solution significantly increased fertilization rates, from 0 to 15%, at similarly low sperm concentrations. It seems therefore that at least in this species, it is possible to enhance fertilization rates at concentrations similar to those existing around the oocyte in the fallopian tube with a technically simple and less aggressive procedure. The potential to treat male infertility by zona thinning was suggested by Kiessling et al. (10). Unfortunately their observations were limited to eight oocytes, of which two fertilized but did not continue to develop following fertilization. It may therefore be that the human oocyte is more sensitive to thinning or, alternatively, that oligospermia may in some instances be associated with dysfunctional sperm incapable of eliciting activation of fertilized oocytes to cleavage. Sperm-zona pellucida recognition and binding are the earliest stage in the interaction between sperm and oocyte. It was found that this binding is mediated by sperm receptors present in the zona pellucida (ZP3) and complementary protein present in the sperm plasma membrane (12). Our demonstration that fertilization occurred in spite of a digested zona pellucida surface (Fig. 1B) suggests that ZP3 exists at a sufficient c o n c e n t r a t i o n throughout the entire thickness of the zona pellucida. The ability of ZP3 to function as a sperm receptor is attributable not to its polypeptide choice but to certain of its oligosaccharides. Pronase, which digests protein, does not affect carbohydrate structure and therefore did not interfere with the binding capacity of sperm. On the other hand, the ability of ZP3 to serve as an acrosome reaction inducer depends on its polypeptide chain (12). Our data suggest that Pronase treatment seems to have spared the capacity of ZP3 to act effectively as both sperm receptor and inducer of the acrosome reaction. In conclusion, this work demonstrates that thinning of the zona pellucida by partial digestion with Pronase is safe and enhances fertilization at very low sperm concentrations. This procedure by itself or in conjunction with other methodologies could Journal o f Assisted Reproduction and Genetics, Vol. 9, No. 2, 1992

ENHANCING IVF BY PARTIAL ZONA PELLUCIDA DIGESTION

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Fig. 1. (A) and (B).

improve the reproductive capacity of men producing sperm with a reduced penetrating ability. ACKNOWLEDGMENT

This research was supported in part by a grant from the Chief Scientist, Israel Ministry of Health. REFERENCES 1. YovichJL, Stanger JD: The limitation ofin vitro fertilization from male with sever oligospermia and abnormal sperm morphology. Vitro Fert Embryo Transfer 1988;1:172

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Journal o f Assisted Reproduction and Genetics, Vol. 9, No. 2, 1992