Mouse t haplotypes are variant forms of chromosome 17 that can be transmitted at non-Mendelian ratios by heterozygous +/t males. The accumulated genetic data indicate that ’ +-sperm’ and ‘t-sperm’ are produced in equal numbers but that most ‘+-sperm’ are rendered dysfunctional, so that ‘t-sperm’ have a relative advan-tage at fertilization. To date, the basis for this t-induced sperm dysfunction has remained unknown. Here we demonstrate that a high proportion of sperm obtained from certain strains of +/t mice undergo a premature acrosome reaction under in vitro capacitation conditions.

The simplest interpretation of these data, in conjunction with previous results, is that developing ‘+-spermatids’ are preprogrammed by ‘i-spermatids’ to undergo this premature reaction. Since acrosome-reacted sperm are unable to participate in the process of fertilization, this defect could account for the extreme distortion of transmission ratio observed from mice heterozygous for a class of complete t haplotypes.

t haplotypes represent a variant form of the proximal portion of chromosome 17 that is found at frequencies of 10-30 % in most wild populations of house mice (see Silver, 1985, and Klein, 1986 for reviews). Although t haplotypes can express deleterious effects such as male sterility or lethality in homozygous individuals, these variant chromosomes are propagated by means of a preferential distortion of transmission ratio from het-erozygous +/t males. In some cases, 99% or more of the offspring produced by +/t males receive the t-carrying homologue of chromosome 17 from their father.

Early workers assumed that transmission ratio distor-tion (TRD) was caused by an actual alteration of the process of chromosome segregation during meiosis. However, studies with cytologically-marked chromo-somes demonstrated that chromosome 17 segregation during meiosis was normal in +/t heterozygous males (Hammerberg &. Klein, 1975). Postmeiotic differen-tiation of germ cells in ±/t males is also morphologi-cally indistinguishable from that observed in wild-type animals (Hillman &. Nadijcka, 1978). Furthermore, equal numbers of ‘+-sperm’ and ‘r-sperm’ are produced and maintained 90min after ejaculation, in the female reproductive tract (Silver &. Olds-Clarke, 1984).

TRD could be explained either by a superiority in fertilizing ability associated with t-sperm, or by a defect in fertilizing ability associated with +-sperm. This question was resolved by artificial insemination exper-iments performed with mixtures of sperm from +/t and +/+ congenic animals (Olds-Clarke &. Peitz, 1985). The results demonstrated that most +-sperm produced by heterozygous males are dysfunctional, whereas t-sperm appear to retain the same fertilizing potential as +-sperm produced by wild-type animals. In vitro ferti-lization experiments, with sperm obtained from hetero-zygotes that carry the highly distorting tw5 haplotype, indicate that ‘+-sperm’ have a reduced ability to fertilize an egg even when all of the barriers normally imposed by the female reproductive tract have been removed (Garside &. Hillman, 1989). Finally, exper-iments with chimeric animals have demonstrated that the dysfunctional state is induced in the meiotic partners of ‘#x2018;t-spermatids’ during the haploid phase of spermatogenesis (Seitz &. Bennett, 1985). To date, the physiological basis for the +-sperm dysfunction has remained unknown, and no protocol has allowed the distinction of +-sperm and t-sperm.

The first phase of the fertilization process is the attachment and binding of sperm to the zona pellucida which encompasses the egg (see Wassarman, 1987 for review). This binding induces the sperm acrosome reaction which allows the sperm to penetrate the zona pellucida into the perivitelline space, where fusion between the sperm and egg plasma membranes can occur. Both binding and induction of the acrosome reaction are mediated by the zona pellucida glyco-protein ZP3. The acrosome is a lysosome-like organelle located in the anterior region of the sperm head above the nucleus and beneath the plasma membrane. The acrosome reaction is an exocytotic event in which the plasma and acrosomal membranes fuse and vesiculate, leading to the release of enzymes that digest the zona proteins (Saling et al. 1979; Saling &. Storey, 1979; Florman &. Storey, 1982; Bleil &. Wassarman, 1985).

Even under normal conditions, some sperm undergo the acrosome reaction prematurely, before coming into contact with the egg (Florman &. Storey, 1982). These acrosome-reacted sperm are unable to bind the zona pellucida, and are effectively nonfunctional (Saling et al. 1979). Here we report that a high proportion of sperm from heterozygous +/t mice undergo the acro-some reaction prematurely during in vitro capacitation. The simplest interpretation of the data is that this population represents the dysfunctional +-sperm.

Mice

All animals were bred in our colony at Princeton University. The tw5 haplotype is present on the congenic 129/SvJ inbred background (N25 or greater for all experiments in this study). The transmission ratio of tw5 in this stock is 98 %. The tlub7 haplotype (Silver et al. 1987) is maintained on a non-inbred background in a balanced lethal genotype with the dominant T mutation (T/tlub7). For this study, T/tlub7 females were crossed with inbred 129/SvJ males to obtain experimental (+/tlub7) and control (+/T) animals from the same litter. The T mutation has no effect on transmission ratio or other aspects of spermatogenesis; thus, +/T animals are functionally wild-type for the purpose of this study. The transmission ratio of tlub7 is 98%.

Sperm collection and in vitro capacitation

Sperm were collected from the caudae epididymides and the vasa deferentia into prewarmed RPMI medium 1640 (GIBCO), supplemented with 24mM bicarbonate, 36μg ml-1 sodium pyruvate, 4 mg ml-1 bovine serum albumin and 25 mm-Hepes buffer. Sperm were either prevented from undergoing the acrosome reaction with the addition of EGTA to 4mM immediately after isolation (Saling et al. 1979), or were allowed to capacitate for one hour at 37 °C (Wolf &. Inoue, 1976). Sperm were pelleted at 2700g for 5min, resuspended in a small volume (0·2-0·5 ml) of supplemented RPMI medium, and fixed overnight with the addition of 8 vol. of phosphate-buffered saline containing 1 % glutaraldehyde.

Slide preparation and microscopy

Fixed sperm samples were washed three times and resus-pended in 0·2-0·4ml of 50mm-triethanolamine. 1-2μ1 of each sample were spotted onto precleaned glass slides and air-dried. Slides were mounted with PBS containing 30% glycerol and 1 % glutaraldehyde, and observed by Nomarski differential interference contrast (DIC) optics (Carl Zeiss, Inc.) using either a 63x Planapo or a lOOx Plan objective.

Statistics

All calculations of significance were performed by chi-squared analysis with one degree of freedom.

Nomarski DIC microscopy can be used reliably to score for the presence or absence of intact acrosomes on mouse sperm. This has been demonstrated by the observation of a strong correlation between the pres-ence of a prominent ridge overlying the anterior region of the sperm head (with DIC optics), and the ability to bind the monoclonal antibody HS19A9 or 125I-ZPS, both of which are indicative of acrosome-intact sperm (Florman et al. 1984; Bleil &. Wassarman, 1986). Acro-some-reacted sperm lack the prominent ridge, and are unable to bind to HS19A9 or 125I-ZP3 above back-ground levels.

We compared the proportions of acrosome-intact and acrosome-reacted sperm obtained from the congenic inbred pair 129/SvJ (129-+/+)and 129-+/tw5, prior to and after capacitation in vitro (Table 1). In most exper-iments, sperm from the cauda epididymes and the vas deferens of individual animals were pooled. However, for males 4-6 of both genotypes (Table 1), epididymal and vas deferens sperm were analyzed separately. Data shown for these animals represent results obtained with epididymal sperm. Acrosome-reacted levels in vas deferens sperm were not significantly different from those observed in epididymal samples either before or after capacitation in any case (data not shown).

Table 1.

Percentage of acrosome-reacted sperm before and after capacitation

Percentage of acrosome-reacted sperm before and after capacitation
Percentage of acrosome-reacted sperm before and after capacitation

Prior to capacitation, acrosome-reacted levels were not significantly different (P>0·50) between sperm from 129-+/ + (30·6% overall) and 129-+/tw5 (31·9% overall; see Fig. 1). After capacitation, the proportion of acrosome-reacted sperm from 129-+/tw5 (67·2% overall) was significantly higher (P< 0·0001) than the proportion from 129-+/ + (34·5 %).

Fig. 1.

A subset of +/t sperm undergo a premature acrosome reaction during in vitro capacitation. The percent acrosome-reacted sperm is shown before (solid bar) and after (hatched bar) one hour of in vitro capacitation. Averages and standard deviations are from Table 1.

Fig. 1.

A subset of +/t sperm undergo a premature acrosome reaction during in vitro capacitation. The percent acrosome-reacted sperm is shown before (solid bar) and after (hatched bar) one hour of in vitro capacitation. Averages and standard deviations are from Table 1.

Similar studies were performed on sperm isolated from males heterozygous for a second highly transmit-ting t haplotype, vu. Acrosome of sperm from six +/tub7 and six wild-type littermates both before and after capacitation are presented in Table 1. Prior to capacitation, sperm from both +/tIub7 and wild-type animals exhibited acrosome-reacted levels similar to those obtained with the congenic pair described above (Table 1). However, after capacitation. the proportion of acrosome-reacted sperm from +/t’ub7 (54·6% over-all) was significantly higher (P< 0·0001) than the pro-portion from wild-type littermates (35·7%).

We have investigated the possibility that a premature acrosome reaction might be involved in the phenotype of transmission ratio distortion expressed by males heterozygous for t haplotypes. A highly significant difference in the proportion of acrosome-reacted sperm present after in vitro capacitation was observed be-tween mice congenic for the twS haplotype. Since the experimental and control animals used in this compari-son were members of a congenic pair, the observed phenotypic difference must be a consequence of genes within or closely linked to the tw5 haplotype. Similar results were obtained in comparisons of experimental and control Fi littermates with or without a second t haplotype, Together, these data argue strongly in favor of the hypothesis that the elevated levels of acrosome-reacted sperm must be caused by genes within the t haplotype itself.

The status of the acrosome has clear functional significance in the fertilization process. Free-swimming, acrosome-reacted sperm are unable to bind to the zona pellucida and thus are incapable of fertilization (Saling &. Storey, 1979; Florman &. Storey, 1982). Background levels of such nonfunctional sperm exist in samples obtained from all mice. In this study, 49·7% of +/tw5 and 29·4% of +/fub7 sperm were found to be acro-some-reacted, after subtraction of background levels obtained from appropriate control animals (Fig. 1). Thus, the presence of the t haplotype has caused 29-50% of the sperm normally available for fertiliz-ation to undergo a premature acrosome reaction. As first suggested by Gluecksohn-Waelsch (1972), trans-mission ratio distortion from +/t heterozygotes is now known to be a consequence of the dysfunction of ‘+-sperm’ that bear the wild-type homologue of chromo-some 17 (Olds-Clarke &. Peitz, 1986; Seitz &. Bennett, 1985). Therefore, the simplest interpretation of the data presented here is that the majority of ‘+sperm’ (58 % from +/tlub7-, greater than 99% from +/tlw5) have become acrosome-reacted and dysfunctional during in vitro capacitation. Since in vitro capacitation mimics the passage of sperm through the female reproductive tract, an analogous effect could occur in vivo.

The alternative explanation of the data is that the presence of a t haplotype in 50 % of the differentiating cells causes (1) the inactivation of up to 50 % of both + and t sperm types by a premature acrosome reaction, and in addition (2) a further inactivation of the remain-ing + sperm by some other mechanism. Although this alternative interpretation cannot be ruled out at the present time, the accumulated data would argue against it.

Studies with chimeric animals have demonstrated that the deleterious effects of t haplotypes are limited to meiotic partners of i-bearing spermatids (Seitz &. Bennett, 1985; Papaioannou et al. 1979). If our first in-terpretation is correct, f-spermatids must preprogram their wild-type meiotic partners during spermiogenesis to undergo the acrosome reaction at a later point, during in vitro capacitation or passage through the female reproductive tract.

The level of transmission ratio expressed by some complete t haplotypes (including and tw32) can be affected by a number of factors such as genetic back-ground (Olds-Clarke &. McCabe, 1982; Bennett et al. 1983; Gummere et al. 1986), time of mating relative to ovulation (Braden, 1958), and in vitro fertilization (McGrath &. Hillman, 1980a,b). With this first class of t haplotypes, transmission ratios can vary from as high as 99 % to as low as 34 %. In contrast, a second class of t haplotypes (exemplified by tw5) is transmitted at a very high ratio (greater than 95 % ) independent of all external factors (Yanagisawa et al. 1961; Garside &. Hillman, 1989; unpublished data). Studies similar to those described here have been conducted with tw32 (Olds-Clarke, 1989) and with (unpublished data), and have failed to show elevated levels of acrosome-reacted sperm. Therefore, transmission ratio distortion can occur from mice heterozygous for the first class of t haplotypes without the induction of a premature acro-some reaction. However, the rapid, premature acro-some reaction observed with +/tw5 and +/tlub7 sperm could explain why the transmission ratio of the second class of t haplotypes remains high under all conditions.

Genetic experiments have identified at least six separate t haplotype loci that play a role in the TRD phenotype (Lyon et al. 1984; Silver &. Remis, 1987; Silver, 1989). Candidate clones for three of these loci have been obtained (Willison et al. 1986; Rappold et al. 1987; Schimenti et al. 1988), and candidates for the products of the other loci have been identified as polypeptide spots within high resolution, two-dimen-sional gel patterns (Silver et al. 1983). Although no information is available concerning the biochemical or physiological effects of the individual products of any of these loci, differences have been observed in compari-sons of whole sperm populations from heterozygous +/t and wild-type +/+ animals. Shur and his col-leagues have demonstrated a higher level of galactosyl-transferase activity in association with sperm from +/t and t/t animals (Shur, 1981; Scully &. Shur, 1988). Olds-Clarke (1989) has demonstrated that +/t sperm undergo a change in swimming behavior (known as hyperactivation) earlier during capacitation than sperm from congenic +/+ animals. Therefore, it seems reasonable to suggest that TRD could result from a combination of several different, but additive, effects of 7-spermatids’ on their meiotic partners. The accumu-lated data suggest that the induction of a programmed, premature acrosome reaction may be one of the factors responsible for the high level of TRD expressed by the second class of t haplotypes described above.

This research was supported by an NIH grant (HD20275) to LMS. JCT was supported by a postdoctoral fellowship from the American Cancer Society.

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