A mouse line bearing a targeted deletion of the Crisp1
gene was generated to elucidate the functional role of epididymal protein CRISP1 in the fertilization process. When Crisp1−/−
males were bred with females of proven fertility, they were found to be fertile. Mating of Crisp1−/−
males with Crisp1−/−
females also showed normal fertility rates indicating that the genetic modification did not affect male or female fertility. This result adds to a growing number of reports of fertile mice lacking proteins proposed to be involved in the fertilization process (Okabe and Cummins, 2007
). Based on the observation that these mice often exhibited other reproductive deficiencies (Baba et al., 2002
; Lu and Shur, 1997
males were evaluated for evidence of changes in various parameters related to reproductive function. No differences compared to controls were observed in the testes or epididymides at histological level and no apparent changes were detected in the morphology or motility of epididymal sperm either before or after capacitation. Because it has been reported that the presence of CRISP1 during rat sperm capacitation inhibits protein tyrosine phosphorylation (Roberts et al., 2003
), we investigated whether the absence of the protein in the mutant mice produced an increase in the levels of this capacitation-dependent event. Capacitated sperm from Crisp1−/−
mice exhibited, however, clearly lower levels of tyrosine phosphorylation than controls, suggesting that CRISP1 might play a regulatory role during mouse sperm capacitation different from that proposed for the rat (Roberts et al., 2006
). An alternative possibility is that the decrease in tyrosine phosphorylation is due to an indirect effect produced by the absence of CRISP1 in the sperm plasma membrane. The molecular mechanism underlying this inhibition is at present under investigation. In spite of the lower levels of tyrosine phosphorylation in Crisp1−/−
mice, no differences were observed in either the spontaneous or progesterone-induced AR when compared to controls. These observations are in agreement with previous reports showing normal levels of AR even in mutant sperm in which tyrosine phosphorylation was completely abolished (Xie et al., 2006
). As far as we know, this is the first report showing a significant decrease in tyrosine phosphorylation accompanied by normal levels of fertility. These results suggest that protein tyrosine phosphorylation is either not required or, required in low levels such as those observed in our study, to achieve spontaneous or progesterone-induced AR or normal fertility.
fertilization experiments showed that Crisp1−/−
sperm were as capable as control sperm of fertilizing cumulus-intact eggs. The normal fertilization levels seen under these conditions might be due to the known beneficial effects of the cumulus matrix for fertilization (Yanagimachi, 1994
). After removal of the cumulus cells, however, Crisp1−/−
sperm exhibited a fertilizing ability significantly lower than controls. Although we cannot exclude the possibility that this inhibition is due to an effect on sperm capacitation (Nolan et al., 2004
; Xie et al., 2006
), the observation that sperm from Crisp1−/−
mice exhibited normal rates of both spontaneous and induced AR, supports an inhibitory effect at the sperm-ZP level. These results are consistent with our recent report proposing a novel role for CRISP1 in sperm-ZP interaction (Busso et al., 2007a
In agreement with the postulated role of rCRISP1 in gamete fusion (Cohen et al., 2000a
; Cohen et al., 2001
; Rochwerger et al., 1992
), in vitro
assays using ZP-free eggs showed a significant reduction in the fusion ability of Crisp1−/−
sperm which became even more evident when mutant sperm were subjected to a competitive fertilization assay. This effect is probably not due to the decrease in protein tyrosine phosphorylation because normal levels of gamete fusion have been reported for sperm that have not undergone this event during capacitation (Nolan et al., 2004
; Xie et al., 2006
). Thus, the overall results of the in vitro
fertilization studies indicate that sperm lacking CRISP1 present a disadvantage in their ability to both interact with the ZP and fuse with the egg.
Despite the compromised sperm fertilizing ability, Crisp1−/−
males were fertile. In this regard, it has been reported that different phenotypes can arise from the same mutation depending on the genetic background. For example, female mice bearing a mutation in the CD81 tetraspanin were fertile when originally derived but showed a reduced fertility after backcrossing onto homogenous background (Rubinstein et al., 2006
). Thus, the possibility that CRISP1 is essential for male fertility in a different genetic background, cannot be ruled out.
Our observations in Crisp1−/−
mice might also be due to other CRISP proteins compensating for the lack of CRISP1. This possibility was explored by examining the inhibitory effect of different CRISP proteins on the fusion ability of Crisp1−/−
sperm. The finding that rCRISP1 and mCRISP2 but not hCRISP1 significantly reduced ZP-free egg penetration by Crisp1−/−
sperm indicates that a CRISP protein with an egg-binding site homologous to CRISP1 might also be involved in gamete fusion. In this regard, recent experiments using an antibody against CRISP2 (which does not cross react with CRISP1) as well as CRISP1 and CRISP2 proteins in competitive studies, revealed the involvement of CRISP2 in mouse gamete fusion through its interaction with the same egg-binding sites than CRISP1 (Busso et al., 2007b
). Together, these results support CRISP2 as a candidate molecule to cooperate with CRISP1 during fertilization. Furthermore, the egg-binding site of mCRISP2 (Signature 2) is only 2 amino acids different from that corresponding to rCRISP1 (Ellerman et al., 2006
), suggesting that CRISP1 and CRISP2 might act through a similar molecular mechanism being capable of compensating for each other. A similar situation has been reported for the tetraspanin CD151 knockout mice in which the lack of the protein could be compensated by BAB22942 (Wright et al., 2004
), a homologous molecule exhibiting differences in only 2 out of the 11 amino acids of the active site (Berditchevski et al., 2001
Another member of the CRISP family, CRISP4, has recently been identified in mouse (Jalkanen et al., 2005
) and rat (Nolan et al., 2006
) epididymides. Although the function of this protein remains unknown, its high homology with hCRISP1 (now considered the human ortholog of rodent CRISP4), opens the possibility that mouse CRISP4 may also be involved in sperm-egg fusion as proposed for hCRISP1 (Cohen et al., 2001
). However, the observation that hCRISP1 (with only 1 amino acid different in its Signature 2 compared to rodent CRISP4), failed to both bind to mouse eggs and inhibit their penetration by Crisp1−/−
sperm does not support CRISP4 as the candidate molecule to compensate for the lack of CRISP1. Nevertheless, it is important to note that the protein compensating for CRISP1 in mutant sperm (i.e. CRISP2, CRISP4 and/or another CRISP) would do so only partially as judged by the significantly impaired fertilizing ability of Crisp1−/−
sperm. The generation of knockout animals for CRISP2 and CRISP4 will provide important information in this regard.
Previous observations from our laboratory showing that immunization of male and female rats with rCRISP1 significantly inhibited fertility in both sexes supported the relevance of CRISP1 for animal fertility and its potential use for fertility regulation (Cohen et al., 2007
). Our present findings do not rule out the possibility of developing a contraceptive approach to block or inhibit CRISP1 protein function in normal adult individuals, a situation clearly different from that corresponding to a knockout animal in which compensatory mechanisms might arise during prenatal or postnatal life.
Together, the results of the present work indicate that CRISP1 is a player in the fertilization system. To our knowledge, this is the first knockout mice generated for a CRISP protein. Continued effort to understand CRISP family function will not only provide a step towards understanding the molecular mechanisms underlying mammalian fertilization but will also provide more information about the versatile capabilities of this growing and evolutionarily conserved protein family.