Localization and role of sulfoglycolipid immobilizing protein 1 on the mouse sperm head.

Sulfoglycolipid immobilizing protein 1 (SLIP 1) is an evolutionally conserved sperm head plasma membrane protein (M(r) = 68 kDa) that binds to sulfogalactosylglycerolipid (SGG), the major sulfoglycolipid present in mammalian sperm. The purpose of this study was to characterize the initial localization and the immunoaggregated relocalization of SLIP1 on the mouse sperm head. Direct immunofluorescence (DF) of live sperm using FITC-antiSLIP1 Fab fragments and FITC-antiSLIP1 IgG indicated that SLIP1 was present in the postacrosomal region of the sperm head, although the intensity of immunostaining by FITC-antiSLIP1 IgG was greatest at the border between the postacrosomal region and the acrosome. Unlike that observed with FITC-antiSLIP1 Fab, DF using FITC-antiSLIP1 IgG indicated that SLIP1 was also present in the anterior tip of the sperm head convex ridge. Results from electron microscopic studies, using antiSLIP1 IgG followed by protein A-gold on live mouse sperm, were similar to the DF findings. In contrast, indirect immunofluorescence (IIF) of live mouse sperm using antiSLIP1 IgG and FITC-secondary antibody IgG detected SLIP1 in the sperm head convex ridge only. The IIF and DF results strongly suggest that these bivalent antibodies could induce the sperm antigen relocalization on live sperm heads. SLIP1 redistribution may be dependent on availability of excess SGG, the SLIP1 binding ligand, based on the observation that purified exogenous biotinylated SLIP1 bound to live mouse sperm at both the postacrosomal and convex ridge regions of the mouse sperm head. Immunoaggregation induced by the primary antiSLIP1 IgG or antiSLIP1 Fab with secondary antibody IgG did not cause the acrosome reaction, suggesting that SLIP1 is not involved in sperm signal transduction. Furthermore, postacrosomal SLIP1 was shown to be involved in zona binding, since sperm pretreated with antiSLIP1 Fab fragments (100 micrograms/ml) bound to the egg zona pellucida in vitro at approximately 35% of control levels.

N-CAM alterations in splotch neural tube defect mouse embryos.

The splotch (Sp) mouse is a model for both neurulation defects and defects in neural crest cell (NCC) derivatives. Since neurulation and NCC emigration from the neural tube occur at similar times in development, we suggest that these two events share a mechanism that, if disrupted, leads to malformations in both developmental pathways. Previous studies have shown that the underlying defect in these mutants may involve a mechanism that alters cellular organization and communication. Cell adhesion molecules (CAMs) have been linked with such interactions and because some, including N-CAM, are involved in neural development, we were interested in their pattern of expression in the splotch mutant. Immunolocalization studies showed similar temporospatial distributions of N-CAM antibody in embryonic day 9 mutants and controls. However, mutant embryos had a much higher intensity of anti-N-CAM fluorescence compared to controls. Further characterization using immunoblot analysis revealed that Sp mutants have an altered N-CAM polypeptide profile. Two N-CAM isoforms (Mr 140K and 180K, K = 10(3] are normally present at this time of development. However, extracts from Sp embryos display a heavier N-CAM species (Mr 200K), as well as an altered 140K isoform. Heterozygotes also exhibit a different N-CAM profile, displaying a band between 180K and 200K in addition to the normal 180K and 140K species. Microheterogeneity was also observed in mutant and heterozygous embryos carrying Spd, an allele of Sp. However, these differences were less dramatic than that of Sp. The Sp locus may be involved in post-translational modification of N-CAM. An aberration in N-CAM processing could be the primary target of the mutation that leads to the development abnormalities observed in this mouse mutant.

Delayed neural crest cell emigration from Sp and Spd mouse neural tube explants.

Splotch (Sp) and splotch-delayed (Spd) are allelic mutations on chromosome 1 of the mouse. Embryos homozygous for either allele have neural tube defects (NTDs) and deficiencies in neural crest cell (NCC) derived structures. The fact that Spd mouse mutants sometimes have deficiencies in NCC derivatives in the absence of an NTD led to the hypothesis that neurulation and the release of NCCs may depend on a regulatory event that is common to both processes. Therefore, it may be possible to understand the cause of NTDs in these mutants by examining the basis of aberrant NCC derivatives. Caudal neural tubes were excised from day 9 Sp and Spd embryos and placed into gelatin-coated tissue culture dishes, or 3-dimensional basement membrane matrigel, and cultured for 72 hours. A cytogenetic marker was used to genotype the embryos. In planar cultures, no morphological differences were observed between NCCs from neural tube explants of Spd mutants compared to those from heterozygous or wild-type embryos. However, there appeared to be a delay in the release of NCCs from the neural tube in both Sp and Spd mutants, which was particularly evident in Sp. After 24 hours in culture, the extent of NCC outgrowth, as well as the number of NCCs emigrating from explanted neural tubes, was significantly lower in Sp and Spd mutant cultures than in controls. No differences were observed in the mitotic indices among cells which had emigrated. By 72 hours, mutant cultures and their non-mutant counterparts were similar in terms of outgrowth, cell number, and migratory capability. After 24 hours in 3-dimensional basement membrane matrigel, cell outgrowth from Sp explants was also significantly less than controls. The pattern of NCC outgrowth in both types of culture conditions indicates a 24 hour delay in mutant cultures compared to controls. This stems from a delay in the release of NCCs from the neural tube, suggesting that the defect lies within the neuroepithelium with respect to the release of NCCs.

Spinal ganglia reduction in the splotch-delayed mouse neural tube defect mutant.

Splotch and splotch-delayed mutants have anomalies in certain neural crest cell derivatives as well as neural tube defects. A genetic marker was used to identify mutant, heterozygote, and wild-type embryos within a litter, which enabled us to make intergenotypic comparisons. Histological studies of the lumbosacral region of day 15 and day 16 embryos indicated that the splotch-delayed mutant had similar but less severe defects in spinal ganglion development than those reported for splotch (Auerbach: Journal of Experimental Zoology 127:305-329, 1954). The ganglia were extensively reduced in size, residual, or missing in the splotch-delayed mutant, whereas in the splotch mutant, they were virtually nonexistent. Paired comparison analyses showed that all mutant embryos had a significant reduction in their volume of lumbosacral spinal ganglia when compared to their heterozygous and/or wild-type littermates. Also, some heterozygotes were found to have spinal ganglia volumes that were significantly reduced when compared to wild-type embryos. The volume of spinal ganglia was not related to the severity of the neural tube defect. In fact, three mutant embryos, which did not exhibit a neural tube defect, had spinal ganglia volumes comparable to or less than those mutants with open neural tube lesions or curly tails. This shows that the formation of abnormal neural crest cell derivatives is not a result of the neural tube closure defect. We hypothesize that the two anomalies observed in these mutants have a common etiological basis.

Retinoic acid-induced selective mortality of splotch-delayed mouse neural tube defect mutants.

The allelic loci splotch (Sp) and splotch-delayed (Spd) cause neural tube defects (NTDs) in mice homozygous for either of these genes. The polymorphic enzyme isocitrate dehydrogenase (Idh-1) in conjunction with a recombination suppressor was used as a genetic marker to identify embryos homozygous for these alleles. A split dose of all-trans retinoic acid (RA) totalling 5.0 mg/kg administered on gestation day 9/15 and 9/18 (days/h) significantly reduced the frequencies of NTD and of mutant genotypes in marked Spd embryos examined on day 16 without significantly increasing the resorption frequency. There was a nonsignificant decrease in the frequencies of NTD and mutant genotypes in embryos examined on day 11 of gestation. Thus, retinoic acid treatment was associated with selective mortality of the homozygous Spd mutants. No evidence of selective mortality was observed in RA-treated Sp embryos.