Gamete Research 22:369-373 (1989)

Failure of Spermatozoa From T/t Mice to Fertilize In Vitro Is Overcome by Zona Dri I li ng Tariq Ahmad, Joanne C. Conover, Martin M. Quigley, Robert L. Collins, Anthony J. Thomas, Jr., and Ralph B.L. Gwatkin

Reproductive and Developmental Biology, Research Institute and Gynecology, The Cleveland Clinic Foundation, Cleveland, Ohio

Failure of epididymal spermatozoa from Tit mutant mice, but not from tit individuals, to fertilize oocytes in vitro was partially overcome by opening a small aperture in the zona pellucida with acidified Tyrode’s solution to permit direct access of the spermatozoon to the vitellus. This study provides a model system to evaluate requirements for successful zona drilling in the treatment of human infertility and further insights into the effects of the t complex on sperm fertility.

Key words: assisted fertilization, t-locus mice, in vitro fertilization, infertility, mouse

INTRODUCTION

By drilling a small aperture through the zona pellucida of the mouse oocyte with acidified Tyrode’s solution, Gordon and Talansky [ 19861 were able to achieve in vitro fertilization with only 1 % of the sperm concentration that otherwise would be required. An increased rate of fertilization after zona-drilling by micromanipulation also has been observed with in vitro matured rat oocytes [Vanderhyden et al., 19881. A direct demonstration that sperm entry occurs through the drilled aperture was provided by the report of Conover and Gwatkin 11 9881, who showed that fertilization would occur in drilled oocytes even when penetration through the zona was blocked by a monoclonal antibody directed against ZP3, the sperm-receptor glycoprotein of the zona pellucida. Since zona drilling, in combination with in vitro fertilization and embryo transfer, may be a means of treating some types of human male infertility, we sought to develop a model using t-mutant mice. In this paper, we show that zona drilling does not overcome the infertility of sperm from infertile twS/tw7l males. However, sperm from T/tws’w7’ males, which carry only one t complex, leading to a

Received August 1, 1988; accepted October 17, 1988.

Address reprint requests to Dr. Ralph B.L. Gwatkin, The Cleveland Clinic Foundation, 1 Clinic Center, 9500 Euclid Avenue, Cleveland, OH 441955240,

0 1989 Alan R. Liss, Inc.

370 Ahmad et al.

reduction in sperm transport [Tessler and Olds-Clarke, 19811 were found to fertilize oocytes only when an opening was made to allow passage of the sperm into their vitelli.

MATERIALS AND METHODS

Titw5 female mice were mated to T/tw7' males to obtain approximately 50% tailless (T/t) and 25% normal-tailed (t/t) offspring. TIT zygotes perish in utero [see McLaren et al., 19751. Epididymal spermatozoa from T/t and t/t males were then compared for the ability to fertilize mature maternal (Tit"') oocytes (drilled or nondrilled).

Oocytes were obtained by the intraperitoneal injection of 5 i.u. PMSG (Sigma Chemical Co., St. Louis, MO) followed 48 h later by 5 i.u. hCG (Sigma). At 12-13 h after hCG, cumulus masses were released from the swollen ampulla of the oviducts into M16 medium [Whittingham, 19711 containing 0.1% hyaluronidase. The cumu- lus-free oocytes were then washed three times in M 16 medium.

Spermatozoa were expressed from the caudae epididymides and vasa deferentia of the T/t and t/t males into IVF medium [see Hogan et al., 19861 supplemented with bovine serum albumin (30 mg BSA/ml, fraction V, Sigma). The sperm suspension was covered with silicon oil (Dow Corning 200 fluid, Dow Corning Corp., Midland, MI) and incubated at 37°C under an atmosphere of water-saturated 5% C02, 5% 02, and 90% N2 for 1.5 h to allow for capacitation.

Mature T/t oocytes (those exhibiting one polar body) were drilled with Narishige micromanipulators (Medical Systems Corp, Greenvale, NY) attached to a Nikon inverted microscope fitted with Hoffman modulation contrast optics (Modu- lation Optics Inc, Greenvale, NY). The holding pipets (1-mm glass capillaries) were hand-pulled over a microburner and fire-polished with a Narishige microforge to yield an outside tip diameter of 70-100 pm and an inside tip diameter of 10-15 pm. The injection pipets were drawn from thin-walled glass capillaries, containing a filament (World Precision Instruments, Inc., New Haven, CT) to aid filling, with a Narishige pipet puller. The injection pipets had an outside tip diameter of approximately 3 pm and were filled with acid Tyrode's solution (pH 2.3). The oocytes were placed at one end of a 200-400-pl rectangular drop of M2 medium [see Hogan et al., 19861 covered with silicon oil in a Petri dish lid. A single oocyte was grasped with the holding pipet, and the injection pipet was brought into the same focal plane. The acid Tyrode's solution was expelled against the zona until a small aperture was made. The drilled oocyte was then moved to the other end of the rectangular drop, and the process was repeated with another oocyte.

Drilled and nondrilled oocytes were placed in 500 pl IVF medium (30 mg BSA/ml), and 50 pl capacitated sperm suspension was added to yield a final concentration of 2-5 x lo6 spermatozoa/ml. After a 4-h incubation, the oocytes were placed in M16 medium. The cultures were returned to the incubator. The proportion of oocytes exhibiting two pronuclei, or cleaving to the two-cell stage, was recorded 9 and 18 h later, respectively.

RESULTS

No fertilization, as shown by failure to form two pronuclei at 9 h incubation, was observed with spermatozoa obtained from the t/t males, even when an aperture

Failure of Spermatozoa From T/t Mice 371

TABLE 1. Effect of Zona Drilling on the Success of In Vitro Fertilization as Judged by the Proportion of Oocytes Forming Two Pronuclei*

Treatment of Genotype oocytes from Titw5 of sperm Proportion (%) with two pn

donor donor Expt 1 Expt 2

2/12 (17%) 5/10 (50%) Zona-drilled T/tW5/W71

o/ 12 (0%) 0112 (0%) 0112 (0%) oi 12 (0%) o/ I2 (0%) 0112 (0%)

twsitw71

tw5,tw71

Not drilled T/tW5/w71

*5 x lo6 sperm/mI.

had been made in the zona pellucida by zona drilling to allow access of the spermatozoa to the vitellus (Table 1). Sperm from the Tit males also failed to fertilize the intact oocytes. However, fertilization did take place with spermatozoa from the T/t males when the zona pellucida of the oocytes had been drilled before sperm addition.

A similar result was obtained with the sperm from the T/t males in experiments in which successful fertilization was assessed by cleavage to the two-cell stage (Table 2). Since parthenogenesis or fragmentation conceivably could have confounded these results, drilled and nondrilled oocytes that were not inseminated were included in these experiments as controls. These controls failed to undergo cleavage.

DISCUSSION

These data show that although spermatozoa from Tit males are fertile in vivo, they may not be so in vitro, under conditions suitable for other strains [Conover and Gwatkin, 19881. The reason for this in vitro infertility is currently under investiga- tion. When an aperture was made in the zona pellucida to allow direct access of the sperm to the vitellus, then fertilization occurred. Spermatozoa from t/t males, however, failed even to fertilize drilled oocytes.

Sperm from t/t males have been reported to exhibit relatively little forward progression [McGrath and Hillman, 19801, and more recently, computer-assisted analysis has revealed that all t/t sperm are nonprogressive [Olds-Clarke, 19861. Our own observations confirm these reports for tw5/tw7’ (data not shown). We did not observe an obvious abnormality in our T/t spelmatozoa, but Olds-Clarke [ 1983a, 19861 has reported that the progressiveness of sperm from mice carrying one t complex is intermediate between normal and t/t sperm, although the proportion that is motile may be the same. T/t sperm also undergo hyperactivation sooner than nonmutant sperm [Olds-Clarke 19871. Thus, drilling a hole in the zona may bypass these defects in T/t sperm, compensating for the reduced progression and/or premature hyperactivation (perhaps followed by a premature acrosome reaction) that may interfere with the sperm’s ability to cross the zona barrier. It is clear that failure to bind to the zona pellucida is not a cause of failure because we observed many sperm of both T/t and t/t bound to oocytes.

Zona drilling was found to be insufficient to allow sperm from t/t males to fertilize in vitro. Such males exhibit complete male sterility and their sperm do not fuse even with zona-free oocytes, although they are able to undergo an acrosome

372 Ahmad et al.

TABLE 2. Effect of Zona Drilling Oocytes From T/tw5 Mice on Their Ability to Develop to the Two-Cell Stage Following Insemination With Spermatozoa From T/tW5’”” Mice*

Experiment 1 Experiment 2 Experiment 3

Total Total Total Treatments oocytes Two-cell Othersd oocytes Two-cell Others‘ oocytes Two-cell Othersd

Zona-drilled, inseminated 20 16 4 6 4 2 6 5 1

Not drilled, inseminated 9 0 2 7 0 0 12 0 3

Zona-drilled, 15 0 0 not inseminated - - -

Not drilled, not inseminated 6 0 1 - 9 0 2

*2 x lo6 sperm/ml. ‘Necrotic or fragmented.

- - -

- -

reaction [McGrath and Hillman, 19801. Because their nonprogressive motility resembles caput rather than cauda sperm, it is possible that tit sperm may be sterile because they do not undergo complete maturation within the epididymis. Since there are no ultrastructural defects unique to sperm carrying one or two t complexes, the sterility of t/t sperm must involve physiological or biochemical defects [Olds-Clarke, 19881.

Galactosyltransferase, thought to mediate gamete recognition by binding to its specific glyconjugate on the zona, is elevated two- to four-fold on the surface of t-bearing compared to normal sperm [Shur and Bennett, 1979; Scully and Shur, 19881. How this is related to their infertility is at present unclear [Macek and Shur, 19881. It is possible that t-mutant sperm have a different responsiveness to Ca2+, as originally suggested by Olds-Clarke [ 1983bl. Experiments are in progress to determine whether altering the Ca2+ concentration of the medium or changing the albumin concentration or the time period allowed for capacitation will permit sperm from t/t males to fertilize and sperm from T/t males to fertilize without the necessity for zona drilling.

ACKNOWLEDGMENTS

The authors would like to thank Dr. Dorothea Bennett, University of Texas, Austin, who arranged for us to receive mating pairs of the t-mutants.

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