We have previously generated and used a series of Apoer2 ICD mutant mouse lines to molecularly define intracellular components of the Reelin signaling pathway. In this study we utilize four of these mutant lines to investigate the involvement of the Apoer2 ICD in selenium uptake both in the brain and testis (). The Apoer2[ex19]
mice constitutively express a splice variant of Apoer2 that contains or lacks exon 19, respectively. Exon 19 encodes a proline rich 59 amino acid sequence within the intracellular domain of the receptor that contains binding sites for the adaptor proteins postsynaptic density protein of 95kDa (PSD95) and JNK interacting proteins (JIPs). This exon is essential in the regulation of synaptic plasticity by the neuronal signaling protein Reelin. Exon 19 functionally couples Apoer2 to N
-aspartate receptors (NMDAR) probably through the adaptor protein PSD95, resulting in the activation of the ionotropic receptors and regulation of NMDAR dependent synaptic plasticity (Beffert et al. 2005
Another Apoer2 ICD mutant line, Apoer2[Stop], contains the deletion of the entire ICD five amino acids carboxyterminal of the NPxY motif. This deletion includes part of exon 18 and all of exons 19 and 20. These mice, along with the Apoer2[Δex19] mice, show an age-dependent loss of corticospinal neurons signifying the importance of exon 19 as a regulator of neurodegeneration, likely through the interaction with JIPs, adaptor proteins involved in the regulation of the JNK signaling pathway (Beffert et al. 2006).
The fourth Apoer2 ICD mutant line, Apoer2[Dab-]
, has a three amino acid mutation in the cytoplasmic ICD just prior to the NPxY motif. Mutation of residues N893E, F894I, and D895G in the cytoplasmic tail of the receptor abrogates binding of the adaptor protein Disabled-1 (Dab1), an obligatory component of the Reelin signaling pathway. By preventing this interaction, the transmission of the Reelin signal through Apoer2 is disrupted. The NPxY motif is involved in the regulation of endocytosis of numerous proteins. Compared to other LDL receptor family members, the endocytosis coefficient of Apoer2 is very low. The three amino acid mutation so close to the NPxY motif affects, but does not completely abrogate, Apoer2 endocytosis (Beffert et al. 2006b
). However, the mutation does severely impair synaptic plasticity further underscoring the importance of Apoer2 in the regulation of NMDA receptor function.
Burk and colleagues have previously shown a profound effect of selenium deficiency on synaptic function and neuronal survival, which is strikingly similar to the defects we have observed in the Apoer2 ICD mutants (Beffert et al. 2005
; Beffert et al. 2006b
; Beffert et al. 2006a
; Peters et al. 2006
; Burk et al. 2007
; Valentine et al. 2008
). This raised the possibility that the phenotypes of our Apoer2 ICD mutant mice might be, at least in part, caused by selenium deficiency and not by the disruption of neuronal signaling pathways. To exclude this possibility, the Apoer2 ICD mutants, along with wild type and Apoer2 KO controls, were fed a diet supplemented with a level of selenium that provides an adequate supply of this micronutrient to wild type mice. After four weeks the animals were euthanized and their brains, testes, and livers were collected for selenium level analysis. Consistent with previous reports, selenium levels were greatly reduced in both the brain () and testis () of Apoer2 KO mice (Hill et al. 2003
; Olson et al. 2007
). By contrast, none of the Apoer2 ICD mutants had significantly lower selenium levels in brain or testis, compared to wild type animals. As a control, selenium levels in the liver, where Apoer2 is not expressed, were comparable across all genotypes (). Although endocytosis in the Apoer2[Dab-]
animals may be affected by the three amino acid mutation, it is not enough to decrease selenium levels in the brain or testis of these mice. We confirmed this conclusion by visualizing the localization of Sepp1 in the testis using immunohistochemistry. The WT animals and all of the Apoer2 ICD mutants showed vesicular Sepp1 staining at the basal lamina (). As previously reported, there were no Sepp1 positive vesicles detected in the Apoer2 KO testes. These data indicate that the domains crucial for regulating synaptic function in the brain are not involved in the uptake of selenium in either of these tissues.
No significant difference in tissue selenium levels of Apoer2 ICD mutant mice from wild type
Vesicular Sepp1 staining observed in all Apoer2 ICD mutant mice
Although none of the Apoer2 ICD mutant mice had significantly decreased selenium levels in the testis, we observed decreased pregnancy rates and small litter size in the Apoer2[Dab-] ICD mutant animals. This impaired fertility in the Apoer2[Dab-] mice indicated a role for Apoer2 in spermatogenesis that is independent of selenium uptake into the testis. Therefore, we investigated the morphology of spermatozoa from wild type and Apoer2 mutant lines by isolating, fixing, and staining spermatozoa with DAPI to label the nucleus, with MitoFluor™ to stain the midpiece, and with a crossreacting polyclonal antibody against an unidentified sperm antigen that marks the acrosome and principal piece of the tail (). The basic organization of the above mentioned sperm sections was identical between the spermatozoa collected from wild type mice and the Apoer2 mutants.
Sperm immunostaining reveals normal organization of spermatozoa in all Apoer2 mutant mice
The Apoer2[Dab-] mice, like the Apoer2 KOs, however, produced a larger fraction of spermatozoa with abnormal tail morphology than wild type mice. We morphologically analyzed spermatozoa from multiple mice (n ≥ 3) from each genotype. Spermatozoa were classified by the shape of the tail as straight, hairpin, angled, or curled (). Straight spermatozoa were defined as those with an absence of a bend in the tail between the neck and the distal tip of the tail. Hairpin spermatozoa were bent back on themselves 180 degrees at the annulus (arrowhead in and ). Angled spermatozoa were also bent at the annulus at an angle between 40-120 degrees with a cytoplasmic droplet often residing at the bend (). The midpieces of curled spermatozoa were looped adjacent to or around the head of the spermatozoon, frequently forming a complete circle. This particular conformation, confirmed by scanning electron microscopy (), appears to coincide with the cytoplasmic droplet failing to migrate down the midpiece and, instead, remaining at the neck. This can cause the head to curl around the droplet. shows the percent of the total population of spermatozoa in each of the four morphological conformations for all six mouse lines. As previously reported, we confirmed that the Apoer2 KO mice produce a considerably higher percentage of hairpin spermatozoa than wild type. The only Apoer2 ICD mutants that significantly differed from wild type with respect to tail morphology were the Apoer2[Dab-] mice. Like the KOs, they had few straight spermatozoa, however, the majority of the Apoer2[Dab-] spermatozoa were split between the hairpin (36.3%±4.1%) and the curled (35.0%±3.1%) conformations. By contrast, 66.3%±2.3% of Apoer2 KO spermatozoa showed the hairpin conformation. This suggests that in the Apoer2[Dab-] mice, migration of the cytoplasmic droplet may be impaired as it remains arrested at the base of the neck in a significant proportion of the spermatozoa, a phenotype not seen in any other Apoer2 ICD mutant line.
Sperm classification into four morphological categories
Quantitative analysis of sperm morphology
This abnormal morphology in the majority of the Apoer2[Dab-] spermatozoa could potentially affect sperm motility, and therefore cause impaired fertility in this mutant strain. To assess any defects in motility, spermatozoa were collected and various established parameters of sperm motility were measured (). Even though there was variability between animals, only the Apoer2 KO mice had a significantly lower percentage of motile spermatozoa compared to wild type. The motility of Apoer2[ex19] spermatozoa was essentially indistinguishable from wild type for all of the parameters tested. By contrast, spermatozoa from the Apoer2 [Δex19] and Apoer2[Stop] lines, both of which lack the alternatively spliced exon 19, had velocities that were considerably reduced compared to wild type but not as much as the KO spermatozoa. This suggests that exon 19, which is essential for synaptic plasticity and neuronal survival, may also play a functional role in sperm motility. However, absence of exon 19 does not reduce motility enough to noticeably impair fertility. Although some of the velocity parameters of the Apoer2[Dab-] spermatozoa were identical to those of the Apoer2 KOs, a larger percentage of the Apoer2[Dab-] spermatozoa were motile compared to the KO mice. Even though fertility is impaired in the Apoer2[Dab-] mice, this modest increase in percent motility may be the factor preventing this Apoer2 ICD mutant line from being infertile like the Apoer2 KOs.
Motility analysis of wild type and Apoer2 mutant sperm