Models for Male Infertility: the T Haplotypes

Reviews of Reproduction (1997) 2, 157–164

Models for male infertility: the t haplotypes

Patricia Olds-Clarke

Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, PA 19140, USA

The t haplotypes are variant alleles of genes in the proximal region of mouse Chromosome 17, linked together by four inversions. While females carrying two t haplotypes are fertile, males are sterile. Their spermatozoa exhibit severe motility defects and are unable to penetrate zona pellucida-free oocytes. Spermatozoa from males carrying one t haplotype (t/+) exhibit mild motility deficits and a delay in penetration of the zona-free oocyte. The inversions of the t haplotypes contain several genes that cause or contribute to male sterility, at least some of which can be identified by analysis of mice carrying Mus spretus–Mus domesticus recombinant Chromosomes 17. The t haplotypes specify a number of sperm biochemical abnormalities, but these have not been related directly to defects in fertilization. In t/+ males, spermatozoa not bearing the t haplotype are defective in fertilization compared with t-bearing spermatozoa. The mechanism causing this is likely to involve haploid gene expression confined to the t-bearing spermatids. Since many genes situated in the region of the t haplotypes have human homologues, an understanding of t haplotype sterility in mice is expected to contribute significantly to our knowledge of the genetic basis for human sperm dysfunction.

The t haplotypes are variant alleles of genes in the proximal Spermatozoa from tx/ty and t/+ males have region of mouse Chromosome 17. Males carrying two t haplo- abnormal motility types are sterile, while males carrying one t haplotype produce two genotypes of spermatozoa, one more fertile than the other. Most spermatozoa from tx/ty males have normal ultrastructure Since there is no evidence that female reproductive processes (Kuretake et al., 1996 and references therein), and the number of are affected by the presence of t haplotypes, t haplotypes must spermatozoa that can be recovered from the cauda epididymis alter events unique to the male. or the uterus is about 50% that of fertile heterozygous (t/+) The t haplotypes were originally called ‘t alleles,’ because and wildtype (+/+) mice (Olds-Clarke, 1986; Olds-Clarke and they were thought to be mutations at a single locus affecting Johnson, 1993). Since mouse sperm counts must fall to below tail development (Silver, 1985). However, it is now clear that 10% of normal before fertilization in vivo is reduced (Searle and t haplotypes encompass the proximal third of Chromosome 17, Beechey, 1974), the modest reduction in numbers of morpho- about 30–40 Mbp of DNA. Furthermore, all t haplotypes con- logically normal spermatozoa observed for tx/ty males cannot tain four large inversions, relative to the wildtype Chromo- explain their sterility. some 17 (Silver, 1985; Hamvas et al., 1996; Fig. 1). In males However, all spermatozoa from tx/ty males have very poor carrying one t haplotype (t/+), these inversions suppress re- motility. The percentage of motile spermatozoa recovered from combination in the region, so that t haplotypes are usually either the epididymis or the uterus is about half that of con- transmitted as a single unit. Thus, t ‘alleles’ are really haplo- genic wildtype spermatozoa. Comparison between congenic types: a particular set of alleles at tightly linked loci. The cor- (that is, genetically identical except in the region of the t com- responding region of wildtype Chromosome 17 is called the plex) mice is very important, because strains of mice differ t complex. markedly in physiological traits important for fertilization Most t haplotypes have lethal mutations at one or more loci (Olds-Clarke, 1988). Movements of most spermatozoa from affecting embryonic development (Barlow, 1992). However, tx/ty males are not recognized by computer-assisted sperm these lethal mutations are located at different sites within motility analysis, so that only the fastest spermatozoa in these various t haplotypes, so that mice carrying two t haplotypes populations can be measured. In these spermatozoa, both with different (complementing) lethal mutations (tx/ty) are curvilinear velocity (an estimate of instantaneous swimming viable. Since males with any two t haplotypes carrying comp- speed) and linearity (an estimate of the straightness of the path) lementing lethal mutations are sterile, all t haplotypes share are reduced by 40%, relative to spermatozoa from congenic common male sterility factors (Lyon, 1991). In this review, the +/+ males. Furthermore, close examination of motile homo- assumption is made that all tx/ty males contain equivalent zygous spermatozoa reveals a unique ﬂagellar curvature, such (noncomplementing) mutations of genes affecting sperm that the ﬂagellum appears coiled or curled in the direction function, and therefore these mice will be referred to as opposite to that of the curvature of the sperm head, referred ‘homozygotes.’ to as ‘curlicue’ (Olds-Clarke, 1986; Olds-Clarke and Johnson, © 1997 Journals of Reproduction and Fertility 1359-6004/97 $10.00 Downloaded from Bioscientifica.com at 09/26/2021 08:58:46PM via free access 158 P. Olds-Clarke

Tctex1 48 119 Cg3-100 122 54 Hba Pim1 Crya 89 (a) Molecular markers

(b) t Haplotype In1 In2 In3 In4 inversions

(d) TRD Tcd1 Tcr Tcd3 Tcd2 factors

(e) Putative Tctex1 Tcte2 Tcp3, Tcp7, Tcp-11 sterility/ Iph1 Tcte3 GalTase activity factor Tcd factors

(f) Hybrid Hst7 Hst4 Hst6 Hst5 sterility factors

Fig. 1. Physical organization of the t haplotypes. (a) The wildtype t complex, which occupies the proximal (to the centromere) one-third of mouse Chromosome 17. The centromere is represented as a ﬁlled circle. Markers used to locate genes or phenotypes within the region are indicated. The markers D17Leh48, D17Leh119, D17Leh122, D17Leh54 and D17Leh89 are represented by 48, 119, 122, 54 and 89, respectively. Distances between markers are not necessarily drawn to scale. Below the wildtype chromosome is the t haplotype form of this region (b), with the inversions (relative to the wildtype sequence of genes) represented by boxes. (c) Locations of the sterility factors, S1–S3; (d) locations of the strongest factors involved in TRD (transmission ratio distortion); (e) location of genes that produce proteins or activities that could be involved in either sterility or TRD; (f) locations of Mus spretus t haplotype hybrid sterility factors. Because M. spretus is inverted relative to M. domesticus in the region of inversion 2, the Hst7 locus could be interrupted by a small segment of wildtype DNA in the proximal region of inversion 2.

1993). Thus, the spermatozoa from the homozygotes have only spontaneous acrosome reaction at rates similar to those of con- weak flagellar force and are not progressive. genic wildtype spermatozoa (Olds-Clarke, 1989; Olds-Clarke and Spermatozoa from t/+ mice also exhibit abnormal motility, Johnson, 1993). These data suggest that t haplotypes decrease but the defects are less severe. Both curvilinear velocity and sperm speed and alter calcium-dependent flagellar curvature linearity are significantly reduced relative to congenic wildtype without greatly affecting other aspects of capacitation. spermatozoa, but are significantly greater than the means for x y spermatozoa from congenic t /t mice. In addition, spermato- Spermatozoa from tx/ty and t/+ males are defective in zoa from heterozygotes display the flagellar curvature and motility-dependent steps in fertilization movement pattern typical of hyperactivated spermatozoa sooner and to a greater extent than congenic wildtype sper- Although more than a million motile spermatozoa from tx/ty matozoa (Olds-Clarke and Johnson, 1993). Furthermore, after mice are deposited in the uterus, few or no spermatozoa are 4 h of incubation, some spermatozoa from heterozygotes also found in the oviduct (Olds-Clarke, 1986 and references therein). exhibit curlicued flagella, although this condition is transitory Thus, the primary cause of sterility in homozygotes is that (Olds-Clarke, 1991). Thus, the spermatozoa from heterozygotes virtually no spermatozoa ever reach the oocytes. Spermatozoa display a less progressive type of movement, relative to wildtype from heterozygotes exhibit reduced transport through the spermatozoa, which appears to be a milder form of the virtually female genital tract (Tessler and Olds-Clarke, 1981). Therefore, nonprogressive motility of spermatozoa from homozygotes. the motility defects caused by t haplotypes are correlated with The motility abnormalities of spermatozoa from heterozygotes decreased or absent sperm transport to the site of fertilization are calcium-dependent (Lindemann et al., 1990), and fewer sper- in vivo. Furthermore, there is direct evidence that it is the less matozoa from homozygotes exhibit a curlicued flagella after in- progressive motility of uterine spermatozoa from hetero- cubation in calcium-poor medium (Olds-Clarke and Johnson, zygotes and homozygotes, and not sperm surface differences, 1993). However, this does not imply abnormalities in other that reduces or eliminates sperm transport to the site of calcium-dependent sperm processes, such as capacitation. fertilization in vivo (Olds-Clarke, 1996). Spermatozoa from t/+ and tx/ty mice become capacitated (as Poor motility could also cause defects in penetration of the measured by the chlortetracycline assay) and undergo the egg investments. Spermatozoa from homozygotes are unable to

Downloaded from Bioscientifica.com at 09/26/2021 08:58:46PM via free access Mouse t haplotypes and male infertility 159 penetrate the oocyte investments, and spermatozoa from rare crossovers occurring within the haplotype, usually between heterozygotes are delayed in reaching the oolemma (Johnson inversions, in t/+ mice. Experiments using these partial t haplo- et al., 1995a). Are their motility abnormalities responsible for types have made it possible to map sterility factors to particular these defects in investment penetration? Sperm penetration of inversions. the oocyte investments depends on many sperm functions as Factors affecting male fertility have been located within well as motility (Yanagimachi, 1988). For example, capacitation inversions 1, 3 and 4 (Fig. 1). The strongest sterility factor, S2, and hyaluronidase activity are thought to play important roles causes sterility when homozygous. Another sterility factor, S1, in cumulus penetration, while spermatozoa must bind to the does not cause sterility by itself, even when homozygous. zona pellucida and undergo an acrosome reaction before zona However, since males homozygous for S1 and heterozygous penetration. However, spermatozoa from both homozygotes and for S2 are sterile (Lyon, 1986), S1 is likely to be a null allele the heterozygotes have a normal rate of capacitation as measured homozygosity of which enhances the detrimental effects of a by the chlortetracycline assay (Olds-Clarke and Johnson, 1993), single copy of S2 (Lyon, 1992). This enhancement effect of S1 is and sperm hyaluronidase concentrations are normal (Erickson also manifest at the cellular level, since spermatozoa from males and Krzanowska, 1974). The ability of spermatozoa from homo- homozygous for S1 and heterozygous for S2 exhibit motility and zygotes to bind to the zona is about half that of spermatozoa zona-free egg penetration defects that are nearly as severe as from congenic +/+ males, but spermatozoa from heterozygotes those of spermatozoa from mice homozygous for two complete bind to the zona to the same extent as congenic wildtype sper- t haplotypes (Johnson et al., 1995b). There is also genetic evi- matozoa. Furthermore, the amount of zona pellucida-induced dence for another, weaker factor, S3, which decreases fertility acrosome reaction of spermatozoa from both heterozygotes and (Lyon, 1986). Thus, the t haplotypes contain mutant alleles at homozygotes is normal (Johnson et al., 1995a). several loci; sperm function is altered by interaction of the Therefore, of the known factors important for penetration of products of these alleles at the cellular or molecular level. the investments, only poor motility and reduced sperm–zona Because of the suppression of recombination between wild- binding appear as possible causes of the inability of spermatozoa type and t haplotype forms of the t complex, identification and from homozygotes to penetrate the zona pellucida. Although isolation of the sterility factors within the inversions has not been the reduction in zona binding could cause a decrease in zona straightforward. Although random cloning from these regions penetration, it seems likely that the complete inability of homo- of the wildtype t complex has produced some interesting candi- zygous spermatozoa to reach the perivitelline space is largely date genes (see below), none of them has been shown to cause due to their extremely poor motility. Since spermatozoa from sperm dysfunction leading to sterility. A novel approach to iden- heterozygotes exhibit no abnormalities except in motility, it tifying t haplotype-related sterility genes has been suggested by appears that even mild motility defects can interfere with pen- studies of Chromosome 17-specific hybrid sterility. etration of the zona. Thus, spermatozoa from tx/ty males appear to be a good model for severe asthenospermia, and Sterility genes in the t complex can be identified by spermatozoa from t/+ mice a good model for mild astheno- analysis of mice carrying Mus spretus–Mus domesticus spermia, uncomplicated by acrosomal defects. recombinant Chromosomes 17

Spermatozoa from tx/ty and t/+ males are defective in When a laboratory mouse (Mus domesticus) carries a t complex from a distantly related species, Mus spretus, together with a fusion with the oocyte t haplotype, the males are sterile. Furthermore, M. spretus alleles Even after removal of the zona pellucida, spermatozoa from at loci within the t complex do not complement t haplotypes homozygotes are unable to penetrate the oocyte (McGrath and with regard to sperm motility (Pilder et al., 1993) or zona-free Hillman, 1980), and spermatozoa from heterozygotes are de- egg penetration (P. Olds-Clarke and S.H. Pilder, unpublished). layed in oocyte penetration (Johnson et al., 1995a). In addition, Since M. spretus sequences within inversions 1, 3 and 4 of the binding to the oolemma does occur, but to a reduced extent t complex are not inverted relative to the wildtype laboratory (Johnson et al., 1995a). Since intracytoplasmic injection of sper- mouse, recombinants of Chromosome 17 of M. spretus and matozoa from homozygotes results in normal blastocysts M. domesticus can be produced whose recombination break- (Kuretake et al., 1996), it appears that the major defect is in fu- points are within inversions 1, 3 and 4. These recombinants can sion with the egg plasma membrane. The best-characterized be used to locate t complex genes affecting male fertility (Pilder candidate for a sperm protein involved in mammalian et al., 1991). With this novel approach, the curlicue ﬂagellum sperm–oocyte binding and fusion is fertilin, but genes for the phenotype has been mapped to a region of less than 1 cM (on two subunits of fertilin are not located within the t complex average, 2 Mbp) within inversion 4 (Pilder et al., 1993). Thus, (Cho et al., 1996). Thus, t haplotypes appear to contain altered using the hybrid sterility model, it is possible to localize genes alleles of a gene or genes, other than fertilin, that are necessary within the t complex that contribute to male sterility. for sperm–egg fusion. t Haplotypes specify molecular defects in t Haplotypes possess several male sterility genes the male germ cells products of which interact Random cloning from the t complex has revealed several gene Evidence that t haplotypes contain more than one gene affect- candidates for sterility factors. Tctex1 speciﬁes a protein that is ing sperm function in fertilization has been provided by studies very similar or identical to a component of cytoplasmic dynein, of partial t haplotypes. Partial t haplotypes are generated by and is found in brain, kidney, liver and spleen, as well as testis

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amino acid change. The protein, TCP11, is found on mature Tcd1 Tcr Tcd3 Tcd2 +/+ epididymal spermatozoa. Fab fragments of an antibody to (a) XXX XXX XX XXXX Tcr transmission >> 50% TCP11 induce premature capacitation and inhibit the spontaneous acrosome reaction in +/+ mice. However, spermatozoa x y Tcd1 Tcd3 Tcd2 from t/+ and t /t males have rates of capacitation and spon- XXX XX XXXX Tcr transmission >> 50% (b) Tcr taneous acrosome reactions similar to congenic +/+ spermato- XXX zoa (Olds-Clarke and Johnson, 1993), suggesting that these Tcr Tcd3 Tcd2 processes are not affected by the t haplotype form of TCP11. (c) XXX XX XXXX Tcr transmission > 50% Nevertheless, it is possible that the t haplotype form of TCP11 alters other sperm characteristics. Tcd1 Tcd3 Tcd2 A series of testicular proteins identified by two-dimensional (d) XXX XX XXXX Tcd transmission = 50% polyacrylamide gel electrophoresis is unique to t haplotype mice. Two, TCP3 and TCP7, are testis-specific and map to inversion 4 (Silver et al., 1987; Fig. 1). However, the function of Tcr (e) XXX Tcr transmission < 50% these proteins is not clear, and it is not known whether they are present in mature spermatozoa. Spermatozoa from tx/ty mice have fourfold higher β-1,4- galactosyltransferase (GalTase) activity, relative to wildtype spermatozoa (Shur, 1981). GalTase has been implicated as an Fig. 2. Interaction of Tcr and Tcd factors in transmission ratio distor- important sperm surface ligand for the zona pellucida protein tion (TRD). Each factor is represented as a series of Xs in the appro- priate region of the t complex. For TRD to occur, Tcr must be ZP3 (Youakim et al., 1994). Since spermatozoa from transgenic present on only one homologue. ‘Tcr transmission’ refers to the per- mice with 20–30 times higher GalTase activity also bind zonae centage of offspring to which the homologue carrying Tcr is trans- poorly, the increased GalTase activity could be responsible for mitted from a male mouse carrying the factors indicated. Tcd factors the reduced zona-binding ability of spermatozoa from homo- have a quantitative effect, regardless of which homologue carries zygotes. Although the GalTase structural gene is not located them (a,b,c). The number of arrowheads refers to the degree of dis- on mouse Chromosome 17, a factor causing increased GalTase tortion, for example, in (a) and (b) transmission is more than 90%, activity is located in inversion 4 (Shur, 1981; Fig. 1). while in (c) transmission is 60 to 85%. Tcr alone also results in TRD, Spermatozoa from tx/ty mice have an abnormal form of but lower than normal (about 20–30%; E). hexokinase. Wildtype mouse spermatozoa have a form of hexokinase phosphorylated on tyrosine residues which appears to be an integral membrane protein located on both the head and (King et al., 1996). There are three single nucleotide differences tail (Visconti et al., 1996). These unusual characteristics suggest between the wildtype and t haplotype forms of Tctex1, and the that this hexokinase could function in sperm–zona or gene co-localizes with S1 (Lader et al., 1989; Fig. 1). This pro- sperm–oocyte binding. Spermatozoa from tx/ty mice have the tein, or something related to it, may also be expressed in plasma membrane-associated form of hexokinase, but it is not mature spermatozoa (O’Neill and Artzt, 1995); even if it is not, tyrosine phosphorylated. The genetic factor responsible for the it could be involved in events important for sperm differen- lack of tyrosine phosphorylation of hexokinase, Iph1, maps to tiation, for example, transport of components to the developing Inversion 1 (Fig. 1; Olds-Clarke et al., 1996). Thus, it is possible axoneme. Alternatively, it could have a function in testicular that the lack of tyrosine phosphorylation of hexokinase is in- cells that is not related to spermiogenesis. volved in the action of sterility factor S1. Another member of the Tctex1 gene family is Tctex2, now While each of these molecular defects in spermatozoa from called tcte3, which maps to inversion 3 (Huw et al., 1995; Fig. 1). tx/ty mice could be involved in a pathway important for fer- The protein appears to be a component of the mouse sperm tail tilization, none have yet been related definitively to sperm (Huw et al., 1995) with homology to a light chain component of dysfunction. Since t haplotypes also carry unique alleles of the Chlamydomonas flagellar outer arm dynein (King et al., 1996). genes expressed strongly in male germ cells, but not directly Since the t haplotype form of tcte3 is different from the wild- related to sperm function in fertilization (for example, Tcp1, type in amino acid sequence and amount of expression (Huw a subunit of the TCP1 ring complex; Kubota et al., 1994), it et al., 1995), it could affect the axonemal function of homozygous will be important to determine whether any of the sperm spermatozoa. However, it is not known whether abnormalities molecular abnormalities of tx/ty mice are involved in their in sperm movement map to inversion 3. sterility. Also located in inversion 3 is Tcte2 (Braidotta and Barlow, in press; Fig. 1). This gene is expressed in wildtype cells during +-Bearing spermatozoa from t/+ mice are defective in meiosis. In t haplotypes, a deletion in the 5’ part of the locus fertilization, relative to t-bearing spermatozoa eliminates Tcte2 expression. Although tx/ty males do not exhibit obvious defects in meiosis, it is possible that Tcte2 expression The two genotypes of spermatozoa produced by t/+ males do during meiosis has a detrimental effect only much later, during not have equal fertilizing ability. Heterozyous males transmit fertilization. the t haplotype to most or all of their offspring, a phenomenon Another gene candidate for a sterility factor is tcp11, which known as transmission ratio distortion (TRD). TRD is unique to maps to inversion 4 (Fraser et al., in press; Fig. 1). The t haplo- males, since heterozygous females transmit the t haplotype type form of the protein differs from the wildtype by one to half of their offspring, as expected according to the laws

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Uterus

Oviduct

t sperm (progressive)

+ sperm (less progressive)

Fig. 3. Hypothesis to explain the unequal fertilizing ability of + and t spermatozoa produced by t/+ mice. Both genotypes of spermatozoa are known to be produced in approximately equal numbers, and both genotypes reach the uterus. However, almost all eggs are fertilized by t spermatozoa. The hypothesis is that + spermatozoa have a less progressive type of motility than t spermatozoa from the same male. Since progressive motility is important for transport from the uterus to the oviduct, and for penetration of the cumulus and zona pellucida, the + spermatozoa are at a disadvantage relative to the t spermatozoa. Therefore, fewer + spermatozoa reach the ampulla, and those that do are delayed in penetration of the investments, so that few + spermatozoa are likely to reach the oocytes before they have been fertilized by t spermatozoa in the same population.

of Mendelian genetics. TRD is an example of meiotic drive, Tcp-10b from the t haplotype did not eliminate TRD (Ewulonu a genetic mechanism exploited by many organisms, including et al., 1996), suggesting that Tcp-10b is not Tcr. fruit ﬂies and yeast (Lyttle, 1993). The Tcd factors could be identical to the factors that cause TRD appears to alter the fertilizing ability of mature sper- sterility in tx/ty males, since the three strongest Tcd factors have matozoa. Heterozygous males produce both t and + spermatids been located in the same three inversions as the three strongest in equal numbers (Hammerberg and Klein, 1975), and both sterility factors, and have the same relative strength of effects genotypes are present in uterine sperm populations after coitus on spermatozoa (Lyon, 1984, 1986). In addition, several lines of (Silver and Olds-Clarke, 1984). Genotyping of blastocysts indi- evidence suggest that t spermatozoa produced by t/+ males do cates that TRD has already occurred by this stage, and there is not have enhanced fertilizing ability, relative to wildtype sper- no evidence for death of embryos at preimplantation stages matozoa from wildtype mice. Rather, the + spermatozoa that (Garside et al., 1991). Thus, TRD appears to occur at fertilization. are meiotic partners of t spermatozoa have impaired fertilizing TRD depends upon two types of factors, Tcr and Tcd (see ability (discussed in Olds-Clarke, 1988). Thus, the evidence Olds-Clarke, 1988 for the rationale for these names). For TRD to suggests that TRD is the result of interaction of factors within occur, there must be only one copy of Tcr. Whichever hom- the t haplotypes, which cause the + spermatozoa to be dysfunc- ologue carries Tcr will be transmitted to the next generation at tional relative to the t spermatozoa. a non-Mendelian ratio. The Tcd factors (for example, Tcd-1, -2, -3) can be on either homologue (Fig. 2a,b). The transmission +-Bearing spermatozoa from t/+ mice could be defective ratio of Tcr is increased as more Tcd factors are added (Lyon, in motility 1984; Fig. 2a,c). Without Tcr, no TRD is observed (Fig. 2d) and when Tcr is present without Tcd factors, the transmission ratio The cellular and molecular bases for the differences in sperm is lower than normal (Dunn and Bennett, 1971; Garside and function between + and t spermatozoa produced by t/+ males Hillman, 1989; Fig. 2e). Thus, TRD appears to be a quantitative are not known. The GalTase activities of + and t spermatozoa phenomenon, involving interaction of several loci within the from t/+ mice are not different (Pratt and Shur, 1993). t complex. Although it was claimed that + spermatozoa from t/+ mice Tcr maps to a non-inverted region between inversions 2 undergo a premature acrosome reaction (Brown et al., 1989), and 3 (Fig. 1). A candidate gene for Tcr, Tcp-10b, is expressed acrosome-reacted spermatozoa were not genotyped. Further- only in the testis, and the form of mRNA encoding the t haplo- more, we found no differences in the timing of the acrosome type is different from the form encoding the wildtype, probably reaction between spermatozoa from +/+ and t/+ mice, using due to alternative splicing. However, targeted mutagenesis of the identical strain and haplotype of mice (Olds-Clarke, 1991).

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Spermatids in t /+ mouse Spermatids in t /t mouse

t + t t

Functional Less functional Non functional

Tcr Tcd1 Tcd2 Tcd3 t alleles

+ alleles

Fig. 4. Distribution of Tcr and Tcd gene products in spermatids from t/+ and tx/ty mice. The products of the wildtype alleles of t haplotype loci involved in transmission ratio distortion (TRD) are indicated by the open symbols, and the products of the t haplotype alleles are indicated by the coloured symbols. Gene products of Tcd factors (triangle, octagon and square) are either produced in the diploid cell or, if syn- thesized after meiosis, are distributed equally to all meiotic partners. The gene product of Tcr (star) is assumed to be produced after meiosis, and to be conﬁned to the cell containing the allele. Note that with regard to products of Tcd factors, t-bearing spermatids produced by t/+ mice are not equivalent to t-bearing spermatids produced by tx/ty mice. It is presumed that the absence of products of wildtype Tcd alleles in the latter is the cause of their inability to function in fertilization.

The hypothesis that the t haplotype form of TCP11 enhances (Lenington and Heisler, 1991), or in vitro fertilization (Garside the fertilizing ability of t spermatozoa (Fraser et al., in press) et al., 1991). These treatments have the potential to reduce or is not consistent with the evidence that t spermatozoa from obviate the barriers the spermatozoa must surmount to reach t/+ mice are no more fertile than spermatozoa from congenic the oocytes, and thus decrease the need for highly progressive +/+ mice (Olds-Clarke, 1988). motility. The hypothesis that + spermatozoa from t/+ mice are It is possible that one or more of the motility defects of less progressive than t spermatozoa is also supported by evi- heterozygote spermatozoa could be unique to + spermatozoa dence that fewer spermatozoa reach the site of fertilization in the population. If + spermatozoa were less progressive than after matings to heterozygotes, and that fewer spermatozoa t spermatozoa in utero, this could impair their transport to the from the heterozygote penetrate the zona during the ﬁrst hour site of fertilization and their ability to penetrate the oocyte in- of insemination in vitro. However, deﬁnitive evidence that vestments, giving t spermatozoa an advantage in fertilization there are differences in motility between + and t spermatozoa (Fig. 3). This hypothesis is consistent with several observations. awaits genotyping of spermatozoa from the oviduct and the First, two-dimensional frequency distributions of curvilinear perivitelline space. velocity and linearity of heterozygote sperm populations support the idea that there may be two populations of motile sper- The mechanism causing the difference between + and matozoa, one more progressive than the other (Olds-Clarke t spermatozoa produced by t/+ mice is likely to involve and Johnson, 1993). Second, a t haplotype with a moderately haploid gene action high TRD shows milder motility defects than another t haplotype with a very high TRD (Olds-Clarke and Johnson, 1993). The unequal fertilizing ability of +- and t-bearing spermatozoa Third, when transmission ratios of t haplotypes are only mod- produced by t/+ males is likely to be the result of interaction of erately high (for example, 0.70 to 0.90) after normal mating, factors causing dysfunction in fertilization (such as Tcd-1, -2, -3) the ratio can often be reduced by ‘delayed mating’ (at or after with Tcr, so that the + spermatozoa are at a disadvantage com- ovulation; Braden and Weiler, 1964), postpartum oestrus pared with the t spermatozoa. The presence of the Tcd products

Downloaded from Bioscientifica.com at 09/26/2021 08:58:46PM via free access Mouse t haplotypes and male infertility 163 could cause a ‘haploinsufficiency’ of the products of the wild- milieu of developing spermatids. Thus, it is likely that an type alleles at these loci during spermatogenesis, and ex- understanding of TRD could provide significant new infor- pression of Tcr might then restore fertility to only those germ mation about mammalian spermiogenesis. cells containing Tcr (Fig. 4). In tx/ty mice, where the products of wildtype Tcd factors are completely lacking, Tcr is insufficient References to support sperm function in fertilization. It seems likely that the Tcd factors are either expressed be- Key references are indicated by asterisks. fore completion of meiosis, or are shared between spermatids, Barlow D (1992) Cloning developmental mutants from the mouse t complex. In Development: The Molecular Genetic Approach pp 394–408 since these factors act either in the trans or cis position (Fig. 2). Eds VEA Russo, S Brody, D Cove, and S Ottolenghi. Springer–Verlag, However, Tcr acts only in the cis position (Lyon, 1984), and Berlin is, therefore, most likely to be expressed after completion of Braden AWH and Weiler H (1964) Transmission ratios at the T-locus in the meiosis and to have its product confined to the spermatids mouse: inter- and intra-male heterogeneity Australian Journal of Biological Sciences 17 – t 921 934 in which it is produced (that is, -bearing cells; Fig. 4). If this Braidotta G and Barlow D Identification of a male meiosis-specific gene, is the case, it would be unusual, since the haploid gene prod- Tcte2, which is differentially spliced in species that form sterile hybrids ucts that have been examined to date are shared between sper- with laboratory mice and deleted in t chromosomes showing meiotic matids resulting from a single meiotic event (Caldwell and drive Developmental Biology (in press) Handel, 1991). Brown J, Cebra-Thomas J, Bleil J, Wassarman P and Silver LM (1989) A premature acrosome reaction is programmed by mouse t haplotypes It is not yet possible to propose a molecular mechanism that during sperm differentiation and could play a role in transmission ratio could account for both TRD of heterozygotes and sterility of distortion Development 106 769–773 homozygotes, because it is not yet certain that sterility factors Caldwell KA and Handel MA (1991) Protamine transcript sharing among are identical to Tcd factors. If they are identical, an understand- post-meiotic spermatids Proceedings of the National Academy of Science USA 88 – ing of the molecular nature of the sterility factors could provide 2407 2411 Cho C, Primakoff P, White JM and Myles D (1996) Chromosomal assign- insight into the nature of Tcd factors. In addition, knowledge of ment of four testis-expressed mouse genes from a new family of trans- the nature of the interaction of S1 and S2 products in spermio- membrane proteins (ADAMs) involved in cell–cell adhesion and fusion genesis and in sperm function could suggest the nature of Tcd Genomics 34 413–417 and Tcr product interactions. 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Islam SD, Pilder SH, Decker CL, Cebra-Thomas JA and Silver LM (1993) t t The human homolog of a candidate mouse complex responder gene: Thus, it is likely that many of the haplotype genes involved conserved motifs and evolution with punctuated equilibria Human in sperm dysfunction have homologues in men (Islam et al., Molecular Genetics 2 2075–2079 1993), and that an understanding of t haplotype sterility in mice *Johnson L, Pilder SH, Bailey JL and Olds-Clarke P (1995a) Sperm from will contribute significantly to our knowledge of the genetic mice carrying one or two t haplotypes are deficient in investment and basis for human sperm dysfunction. oocyte penetration Developmental Biology 168 138–149 Johnson L, Pilder SH and Olds-Clarke P (1995b) The cellular basis for inter- However, the mechanisms underlying TRD remain a action of sterility factors in the mouse t haplotype Genetic Research “… huge black box filled with unknowns” (Gluecksohn- (Cambridge) 66 189–193 Waelsch, 1989). 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