We have reported here the creation and extensive morphological, biochemical, and functional characterization of mice lacking a spermatid-specific ubiquitin-conjugating enzyme. Adult knockout mice were fertile and showed normal testes weights, spermatid number, protein content, and rates of ubiquitination. In vitro analyses of sperm showed no evident defects in motility compared to sperm from wild-type mice. Even when subjected to the heat stress of cryptorchidism, the temporal profile of loss of germ cells was not significantly different from wild-type mice.
However, the knockout mice did manifest a subtle delay in postnatal development during the first wave of spermatogenesis. This developmental defect was apparent at days 40 and 45 of life but resolved by day 65. Although this defect was subtle, it appears to be a real finding. Large numbers of mice derived from crosses of different heterozygous parents over a period of 2 years were analyzed at these time points to render it very unlikely that these findings were spurious. Furthermore, heterozygous mice showed a defect intermediate between wild-type and mutant mice, a finding consistent with a gene dosage effect. Although differences were statistically significant only at days 40 and 45, it should be noted that at day 35 the mean of the knockout group was also less than the mean of the wild-type group, but the P value of the difference was borderline (0.06). The likelihood that differences would occur by chance in three consecutive time points when UBC4-testis is normally expressed in wild-type mice would be very remote.
These findings indicate a role for UBC4-testis in regulating testis maturation. This is consistent with our previous finding that UBC4-testis is induced in round spermatids beginning at day 25 of life (27
). However, in the adult testis, there was no evidence of abnormal distribution of germ cell types and in particular no prolongation of duration in the evolution of round into elongating spermatids, as might be revealed by a loss of the normal pattern of cells seen at each stage of spermatogenesis (Fig. ). This shows that UBC4-testis is not required per se for spermatogenesis. Instead, it may have a specific role in promoting the evolution of the first wave of spermatogenesis. Due to the small differences between the testes of wild-type and knockout mice, it has been very difficult to explore further the cellular and biochemical bases for the differences.
Alternatively, UBC4-testis may play a role in regulating the postnatal growth of the testis. Possibly, during the first wave of spermatogenesis, UBC4-testis positively regulates the function of a specific factor that promotes the growth of the testis or it negatively regulates an inhibitor of such evolution. Presumably, UBC4-testis does so by interacting with specific ubiquitin protein ligases that preferably interacts with it as opposed to other UBC4 isoforms. The catch up to the normal size with time suggests that other UBC4 isoforms may interact weakly with this ubiquitin protein ligase(s) and therefore be less effective at mediating ubiquitination with these putative ligase(s). However, over time, an equivalent amount of ubiquitination is completed and a catch up in maturation occurs. Up to now we have not been able to identify any such UBC4-testis specific ligases. We have only identified some ligases, such as ARNIP/Pirh2 (3
) and SCFβ-TRCP
(Y. Ben-Neriah and S. S. Wing, unpublished observations), which function with the ubiquitous UBC4-1 but not UBC4-testis. Therefore, we cannot rule out at this time that these differences are due to a gene dosage effect occurring in isoforms with redundant functions. Overall, UBC4 levels do increase during the elongation of spermatids (20
) and so by mass action could increase ability to bind and support weaker interacting ligases or simply replace the loss of the UBC4-testis isoform. These other UBC4 isoforms are highly similar to UBC4-testis with 91 to 93% amino acid identity so such overlapping or replacement function would not be surprising.
Taken together, our biochemical and genetic data support the concept that highly similar isoforms are not necessarily redundant. For example, markedly reduced expression of UBCM4 (the mouse ortholog of human UBCH7), an isoform more distantly related to UBC4/UBC5, leads to a defect in implantation of the embryo (9
). Similarly, there are two isoforms of UBC2 (HHR6A and HHR6B) that are 96% amino acid identical (14
). They show ubiquitous expression in all cells studied to date, and there is no evidence of any differences in biochemical function between these two isoforms. However, when HHR6B is inactivated in the mouse, it leads to male infertility due to incomplete spermatogenesis (22
). The mutant mice manifest a heterogeneous population of abnormal spermatids. Interestingly, inactivation of the HHR6A isoform in the mouse does not lead to male infertility but female infertility due to an apparent defect in progression of embryos past the two cell stage (21
). Although these results could be due to gene dosage effects due to relatively high expression of HHR6A in oocytes and HHR6B in spermatids, these findings could also be due to specific functions of the two isoforms. In support of this latter possibility, the putative ligase cyclophilin CYC4/hCyP-60 requires HHR6B but not HHR6A (10
), whereas another RING ligase, Rfpl4, interacts with HHR6A but apparently not HHR6B in a yeast two-hybrid screen (26
With the availability of sequences of various genomes, it is clear now that for several of the yeast ubiquitin-conjugating enzymes such as UBC2 and UBC4/UBC5, there are multiple orthologous genes in the mammalian genomes. Our data suggest that these isoforms can serve redundant complementary functions but, in addition, may also have specific roles that help meet the more complex functional requirements of higher organisms.