Oligoasthenoteratozoospermia is one of the most common causes of male subfertility in human populations [19
]. Disruption of the gene encoding the testis-expressed τCstF-64 polyadenylation protein, Cstf2t
, in mice results in oligoasthenoteratozoospermia [17
sperm cells display a very poor motility, and sperm morphology is extremely abnormal, potentially explaining the infertility of Cstf2t−/−
males. Various mouse models of infertility have been produced using gene knockout technology. In each of these models, spermatozoa were often found in the epididymal fluid, and the phenotype of these mice was rarely a complete block of fertilization [36
]. However, only a few mouse models display both abnormal sperm morphology and impaired progressive motility.
For instance, Cnot7
- and Tex18
-null mice represent these kinds of deficient sperm population [38
], but both have produced offspring during breeding experiments. In contrast, no offspring have ever been sired by Cstf2t−/−
Consistent with the complete block of in vivo fertility in Cstf2t−/−
males, the present study is, to our knowledge, the first to demonstrate that a gene involved in the regulation of gene expression and polyadenylation can have a direct effect on sperm-ZP adhesion. In this circumstance, it seems likely that more than one gene product involved in ZP adhesion may be affected by the lack of polyadenylation activity involving τCstF-64.
Although τCstF-64 is expressed in other tissues [6
], the phenotype of Cstf2t−/−
mice is restricted to male germ cells; to date, we have seen no phenotype in any other tissue or in females [17
] (data not shown). This observation suggests that the primary function of τCstF-64 is to support polyadenylation during spermatogenesis and that its role elsewhere is redundant. Interestingly, in male germ cells, Cstf2ttm1Ccma
showed variable expressivity, with many cells displaying defects in elongating and mature spermatids but with a few motile spermatozoa present in the caudae epididymides. This amalgam of cells reflects the various defects in spermatogenesis produced by loss of τCstF-64, and it is reminiscent of oligoasthenoteratozoospermia, an infertility condition diagnosed in the human population.
In the present study, Cstf2t−/−
spermatozoa had a number of head defects but showed progressive motility for some cells. This last observation raised the question of whether assisted reproductive techniques might allow fertilization from this motile population. Upon testing, Cstf2t−/−
spermatozoa enriched for motile cells failed to produce fertilized eggs in vitro, demonstrating that the infertile phenotype of the Cstf2t−/−
spermatozoa in vitro could be attributed not only to low sperm count but also to failure of gamete interactions. Incubation of COCs with Cstf2t−/−
sperm cells resulted in normal dispersion of cumulus cells, indicating that Cstf2t−/−
sperm cells expressed hyaluronidase genes. Six hyaluronidase-like genes have been determined in the mouse genome, and in regions syntenic with the human genome [41
]. Recently, arylsulfatase A, a protein previously reported to have a role in sperm-ZP adhesion [42
], was shown to be involved in cumulus-cell dispersion [43
]. Arylsulfatase A and other components necessary for this action, such as hyal5 and SPAM1/PH-20, were more likely to be moderately affected by the lack of polyadenylation via τCstF-64. Therefore, the block to fertilization seen in Cstf2t−/−
spermatozoa was not at this early step.
The structure and morphology of Cstf2t−/−
spermatozoa are markedly abnormal. Therefore, it is possible that at least part of the block to fertilization is caused by structural defects in these cells. Localization of zonadhesin, proacrosin, SPAM1/PH-20, CST8 (CRES), and ZP3R/sp56 in the apical head was consistent with the results of previous studies in which they were shown to be present in the acrosomal matrix of wild-type mice [4
]. Although sperm head morphology was mostly incorrect within Cstf2t−/−
spermatozoa, each antibody revealed specific proteins colocalized with PNA staining in the crescent shape associated with an intact acrosome, thus failing to identify mislocalization or absence of any of the proteins tested. However, CST8 (CRES) was shown to be associated with the acrosomal matrix of Cstf2t−/−
; an additional subpopulation was detected at the postacrosomal region for certain sperm cells, as shown in B. Moreover, with the exception of CST8 (CRES), each of the acrosomal proteins examined (zonadhesin, acrosin, SPAM1/PH-20, and ZP3R/sp56) in Cstf2t+/+
spermatozoa were previously shown to interact directly with ZP glycoproteins. Nevertheless, CST8 (CRES) is believed to play an important role in the regulation of protein processing, because it has been shown to inhibit prohormone convertase (PC) activity, suggesting a role in proprotein processing [46
]. Certain PCs are exclusively found in the testis. Therefore, PC4 is a sperm-specific molecule [48
], and the lack of this protein produced a severe subfertility phenotype in the absence of sperm defect or impaired motility associated to these null spermatozoa [49
]. Thus, the absence or mislocalization of acrosomal components tested was inconclusive for the failure of Cstf2t−/−
These localization results imply that the presence of acrosomal proteins are perhaps less critical than their processing or their posttranslational modifications during spermiogenesis or fertilization. One or several of the acrosomal molecules could be localized correctly yet be completely nonfunctional as a consequence of aberrant processing. Zonadhesin, a sperm-ZP adhesion molecule that confers species-specificity during fertilization, is a target of proteolytic enzymes during the acrosome reaction (Tardif et al., unpublished results). ZP3R/sp56 is another acrosomal protein that was shown to be a target for sperm proteolytic enzyme but was dramatically reduced in noncapacitating conditions [50
]. The impaired ZP adhesion in Cstf2t−/−
spermatozoa could also be related to defective biosynthesis, processing, or trafficking of other acrosomal molecules. Additional characterization will be necessary to determine whether the mature forms of different acrosomal proteins are correctly synthesized and completely functional in the acrosomal matrix of Cstf2t−/−
Finally, characterization of the round cells observed prominently in the Cstf2t−/− mice suggests these cells contained developmental remnants of earlier spermatogenic stages. These micrographic images suggest problems in tail development, including, but not limited to, incorrectly formed radial spokes and nexin links. Therefore, the round cells may have at least three possible origins: 1) round spermatids that sloughed into the seminiferous tubule lumen prematurely, 2) incorrectly developed spermatozoa that lost tails or had the tails coil inside, and 3) empty cells, the contents of which were lost because of a loss of structural integrity, cell permeability, or osmotic selectivity.
Because polyadenylation is essential for gene expression, it was perhaps not surprising that the germ cell-expressed τCstF-64 proved to be important for spermatogenesis. However, it was surprising that targeted deletion of Cstf2t
resulted in germ cell development that was impeded but not halted. The present results indicate that loss of τCstF-64 results in a large number of structural, biochemical, and developmental defects during spermatogenesis in Cstf2t−/−
mice, probably because of both large and small changes in the expression of multiple genes rather than an absolute block. This hypothesis is consistent with earlier microarray results for testes from Cstf2t−/−
mice in which levels of many mRNAs were altered but not eliminated [17
]. Overall, we believe these changes in gene expression will be important for understanding the mechanisms of polyadenylation as well as the intricacies of spermatogenesis.