As a result of ongoing research into the mechanism of fertilization using gene-manipulated animals, a new scheme is emerging to replace the old models (Ikawa et al., 2010
). Detailed analysis revealed that in at least seven gene-KO mouse lines (for Clgn
, and Adam2
), ADAM3 became undetectable (or showed aberrant localization) in spermatozoa and the male mice were infertile (Cho et al., 1998
; Nishimura et al., 2004
; Yamaguchi et al., 2006
; Ikawa et al., 2011
; Marcello et al., 2011
; Tokuhiro et al., 2012
). Therefore ADAM3 is considered to be a key molecule for ensuring fertility.
Even though they show vigorous movement, when they come into contact with zona pellucida WT spermatozoa stick to it very efficiently. All of the various kinds of ADAM3-deficient spermatozoa were shown to fail in binding to the zona pellucida of cumulus-free eggs. If we assume that ADAM3 is directly involved in sperm–zona binding, then quantifying ADAM3 on the sperm surface is of interest. The presence of ADAM3 on spermatozoa has been verified by Western blotting and immunostaining (Kim et al., 2004
). However, as far as we know, ADAM3 has never been detected by a direct staining method on gels such as by silver staining or by proteomic analysis using mass spectrometry. We hypothesized that another molecule(s) might be more abundant than ADAM3 and function directly in sperm–egg binding. On the basis of this hypothesis, we performed a proteomic analysis of mouse sperm extracts and discovered the possible involvement of Pmis1
in the fertilization process. Here we described the cloning of these genes and their disruption by homologous recombination. Pmis1
was shown not to be essential for fertilization (Supplemental Figure S4), but Pmis2
(RIKEN cDNA 4930479M11), registered as a noncoding gene, was found to be producing mRNA, and the PMIS2 protein was essential for fertilization.
Because PMIS2 became undetectable in sperm by disruption of Clgn or Calr3, we assumed that these chaperones are necessary to fold PMIS2 properly, but we could not demonstrate the interaction of PMIS2 with these molecules by immunoprecipitation. We also tried to identify the localization of PMIS2 on spermatozoa, but in spite of multiple trials (e.g., the use of whole recombinant PMIS2 protein expressed in mammalian cells as an antigen), we were not able to obtain an antibody that worked for immunostaining of native PMIS2 or recombinant PMIS2 on spermatozoa. Therefore the interactions of PMIS2 with other molecules and the localization of PMIS2 on the spermatozoon need to be clarified to understand the function of PMIS2 in fertilization.
Spermatozoa from Pmis2-
KO mice showed normal fertilizing ability in an IVF system with cumulus-enclosed eggs, despite their impaired zona-binding ability. We do not know why; however, this phenomenon was already reported in spermatozoa lacking ADAM3 (Nishimura et al., 2004
; Tokuhiro et al., 2012
). In spite of the normal fertilizing ability in IVF, Pmis2
-disrupted males were infertile in natural mating. The spermatozoa from Pmis2−/−
mice had a deficiency in sperm migration into the oviduct. This indicates that the immediate cause of Pmis2−/−
male infertility is the inability of spermatozoa to reach the oviduct rather than impaired zona-binding ability. Therefore the sperm–zona binding ability measured in vitro using cumulus-free eggs might not be a good indicator of the ultimate fertilizing ability of spermatozoa.
PMIS2 consists of two hydrophobic regions and a putative N
-glycosylation site and shows homology to CD225 (also known as interferon-induced transmembrane protein 1) superfamily, based on motif searching. CD225 has been reported to mediate antiproliferative activity and cell–cell adhesion in human B cells and leukocytes (Deblandre et al., 1995
). If PMIS2 is related to the interaction of spermatozoa with the zona pellucida or with the epithelial cells of the UTJ, a common motif (amino acid sequence GAP to ILV) might be the responsible domain.
We found that PMIS2 was absent from spermatozoa in three independent mouse KO lines, for Adam3, Clgn, and Calr3. From its speculated abundance on spermatozoa, we assume that PMIS2 could be the ultimate factor involved in sperm migration into the oviduct, possibly based on an interaction of the sperm surface with the UTJ epithelium. However, when we disrupted Pmis2, ADAM3 became undetectable in the spermatozoa. Thus PMIS2 is the seventh gene discovered that is essential for ADAM3 to be delivered to the sperm surface. On the other hand, the disruption of Pmis2 did not affect any of the remaining four gene products (CLGN, CALR3, ADAM1a, and ADAM2). Because both PMIS2 and ADAM3 become undetectable in spermatozoa if the other is not expressed, we assume that these proteins might interact closely. However, we have not been able to demonstrate the formation of heterodimers or direct interactions between the two proteins. The identification of PMIS2 as a novel candidate factor for the ultimate sperm–UTJ interaction (and also sperm–zona binding) will help clarify the general mechanism of mammalian fertilization.