Haprin-deficient spermatozoa are incapable of in vitro fertilization.

Haprin (TRIM36) is a ubiquitin-protein ligase that mediates ubiquitination and subsequent proteasomal degradation of target proteins. It is expressed in the testes in both mice and humans and is thought to be involved in spermiogenesis, the acrosome reaction, and fertilization. However, the functional role of Haprin is poorly understood. The aim of this study was to investigate the physiological role of Haprin in fertility. Homozygous haprin-deficient mice were generated and these mice, and their spermatozoa, were analyzed to detect morphological and fertility-related abnormalities. In these models, normal spermatogenesis was observed but sperm quality was reduced with haprin-deficient mice having poorer sperm morphology and motility than wild-type mice. Interestingly, haprin-deficient mice showed normal in vivo fertility but could not fertilize oocytes under standard in vitro fertilization conditions. In conclusion, this study demonstrated that Haprin deficiency causes morphological abnormalities in spermatozoa, indicating that Haprin is involved in spermiogenesis.

Production of mouse pups from germline transmission-failed knockout chimeras.

Occasionally, chimeras do not transmit the gene of interest to pups in gene disruption experiments. However, the risk of failure could be reduced if we could identify embryonic stem (ES)-derived germ cells in the testis. Here, we report the production of pups from three lines of infertile chimeric male mice and the establishment of knockout lines by combining green fluorescent protein-tagged ES cells with intracytoplasmic sperm injection.

Sperm equatorial segment protein 1, SPESP1, is required for fully fertile sperm in mouse.

Mammalian fertilization is a multistep process that culminates in the fusion of the sperm and egg plasma membrane. It is widely accepted that the equatorial segment of the acrosome-reacted sperm is important in initiating fusion with the egg plasma membrane during fertilization. There are various proteins known to be distributed only in the equatorial segment of sperm. The role of these proteins must be clarified to understand the membrane fusion process. We produced a mouse line that lacked SPESP1 (sperm equatorial segment protein 1) and analyzed the fertilizing ability of the sperm. The average number of pups that were fathered by Spesp1(+/-) and Spesp1(-/-) males was significantly lower than that of wild-type fathers. In these mouse lines, fewer sperm were found to migrate into oviducts and fewer eggs were fertilized. The Spesp1(+/-) and Spesp1(-/-) sperm showed a lower fusing ability compared with the wild-type sperm. The disruption of Spesp1 was shown to cause an aberrant distribution of various sperm proteins. Moreover, scanning electron microscopy revealed that the membrane in the equatorial segment area, which usually forms an acrosomal sheath, disappears after acrosome reaction in Spesp1-deficient mice. It was demonstrated that SPESP1 is necessary to produce the fully 'fusion competent' sperm.

Formation of a thymus from rat ES cells in xenogeneic nude mouse↔rat ES chimeras.

Various conditions for differentiating embryonic stem (ES) cells or induced pluripotent stem (iPS) cells into specific kinds of cell lines are under intensive investigation. However, the production of a functional organ with a three-dimensional structure from ES or iPS cells is difficult to achieve in vitro. In the present paper, we describe the establishment of a green fluorescent protein-expressing rat ES cell line and production of mouse↔rat ES chimera by injecting rat ES cells into mouse blastocysts. The rat ES cells contributed to various organs in the chimera, including germ cells. When we injected ES cells into blastocysts of nu/nu mice lacking a thymus, the resultant chimeras produced thymus derived from rat ES cells in their bodies. The chimeric animals may provide a method for the derivation of various organs from ES or iPS cells.

The X-linked imprinted gene family Fthl17 shows predominantly female expression following the two-cell stage in mouse embryos.

Differences between male and female mammals are initiated by embryonic differentiation of the gonad into either a testis or an ovary. However, this may not be the sole determinant. There are reports that embryonic sex differentiation might precede and be independent of gonadal differentiation, but there is little molecular biological evidence for this. To test for sex differences in early-stage embryos, we separated male and female blastocysts using newly developed non-invasive sexing methods for transgenic mice expressing green fluorescent protein and compared the gene-expression patterns. From this screening, we found that the Fthl17 (ferritin, heavy polypeptide-like 17) family of genes was predominantly expressed in female blastocysts. This comprises seven genes that cluster on the X chromosome. Expression analysis based on DNA polymorphisms revealed that these genes are imprinted and expressed from the paternal X chromosome as early as the two-cell stage. Thus, by the time zygotic genome activation starts there are already differences in gene expression between male and female mouse embryos. This discovery will be important for the study of early sex differentiation, as clearly these differences arise before gonadal differentiation.

Comparison of gene expression in male and female mouse blastocysts revealed imprinting of the X-linked gene, Rhox5/Pem, at preimplantation stages.

Mammalian male preimplantation embryos develop more quickly than females . Using enhanced green fluorescent protein (EGFP)-tagged X chromosomes to identify the sex of the embryos, we compared gene expression patterns between male and female mouse blastocysts by DNA microarray. We detected nearly 600 genes with statistically significant sex-linked expression; most differed by 2-fold or less. Of 11 genes showing greater than 2.5-fold differences, four were expressed exclusively or nearly exclusively sex dependently. Two genes (Dby and Eif2s3y) were mapped to the Y chromosome and were expressed in male blastocysts. The remaining two (Rhox5/Pem and Xist) were mapped to the X chromosome and were predominantly expressed in female blastocysts. Moreover, Rhox5/Pem was expressed predominantly from the paternally inherited X chromosome, indicating sex differences in early epigenetic gene regulation.

Mice deficient in the axonemal protein Tektin-t exhibit male infertility and immotile-cilium syndrome due to impaired inner arm dynein function.

The haploid germ cell-specific Tektin-t protein is a member of the Tektin family of proteins that form filaments in flagellar, ciliary, and axonemal microtubules. To investigate the physiological role of Tektin-t, we generated mice with a mutation in the tektin-t gene. The homozygous mutant males were infertile, while the females were fully fertile. Sperm morphology and function were abnormal, with frequent bending of the sperm flagella and marked defects in motility. In vitro fertilization assays showed that the defective spermatozoa were able to fertilize eggs. Electron microscopic examination showed that the dynein inner arm structure was disrupted in the sperm flagella of tektin-t-deficient mice. Furthermore, homozygous mutant mice had functionally defective tracheal cilia, as evidenced by altered dynein arm morphology. These results indicate that Tektin-t participates in dynein inner arm formation or attachment and that the loss of Tektin-t results in impaired motility of both flagella and cilia. Therefore, the tektin-t gene is one of the causal genes for immotile-cilium syndrome/primary ciliary dyskinesia.

Spontaneous ocular surface inflammation and goblet cell disappearance in I kappa B zeta gene-disrupted mice.

PURPOSE: The ocular surface epithelium is part of the mucosal defense system. Because transcription factor NF-kappa B in mucosal epithelial cells plays a central role in regulating the genes that govern the onset of mucosal inflammatory responses, we examined the role of a regulator of NF-kappa B, I kappa B zeta, in murine ocular surface inflammation. METHODS: The eyes of I kappa B zeta(-/-) mice were analyzed biomicroscopically and histologically. I kappa B zeta expression in normal mouse cornea and conjunctiva was examined by RT-PCR. The results were compared with those obtained in other tissues by real-time PCR. I kappa B zeta mRNA on the ocular surface and in other mucosal tissues was localized by in situ hybridization. RESULTS: I kappa B zeta(-/-) mice manifested chronic inflammation, specifically in the ocular surface, but not in other tissues. In normal mice, I kappa B zeta was expressed in a variety of mucosal tissues. The I kappa B zeta transcript was predominantly distributed in the epithelia of these tissues. As inflammatory symptoms progressed on the ocular surface of I kappa B zeta(-/-) mice, inflammatory cells, mainly CD45R/B220(+) and CD4(+) cells, intensely infiltrated the submucosa of the conjunctival epithelia. This infiltration was accompanied by an almost complete loss of goblet cells in the conjunctival epithelia. CONCLUSIONS: The authors postulate that I kappa B zeta in the ocular surface epithelia negatively regulates the pathologic progression of ocular surface inflammation.

Small interfering RNA and gene silencing in transgenic mice and rats.

After short duplexes of synthetic 21-23 nt RNAs (siRNA) were reported to be effective in silencing specific genes, a vector-based approach for siRNAs was demonstrated in mammalian cultured cell lines. However, the effect of RNA interference (RNAi) on various differentiated cells in live animals remains unknown. In this report, we demonstrate that transgenically supplied siRNA can silence ubiquitously expressed enhanced green fluorescent protein in every part of the mouse and rat body. These results suggest that transgenic RNAi could function as an alternative method of gene silencing by applying homologous recombination to embryonic stem (ES) cells, and should be successful even in species where ES cell lines remain unestablished.