Maintenance of genomic methylation patterns in mammalian somatic cells depends on DNA methyltransferase-1 (Dnmt1). Mouse oocytes and preimplantation embryos lack Dnmt1 but express a variant of this protein called Dnmt1o. We eliminated Dnmt1o by deletion of the oocyte-specific promoter and first exon from the Dnmt1 locus. Homozygous animals were normal, but most heterozygous fetuses of homozygous females died during the last third of gestation. Although genomic methylation patterns were established normally in Dnmt1o-deficient oocytes, embryos derived from such oocytes showed a loss of allele-specific expression and methylation at certain imprinted loci. Transient nuclear localization of Dnmt1o in 8-cell embryos suggests that this variant of Dnmt1 provides maintenance methyltransferase activity specifically at imprinted loci during the fourth embryonic S phase.

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The acquisition of genomic methylation in the male germ line is initiated prenatally in diploid gonocytes, while DNA methylation in the female germ line is initiated postnatally in growing oocytes. We compared the temporal expression patterns of the DNA methyltransferases, DNMT1, DNMT3a, DNMT3b, and DNMT3l in the male and female germ lines. DNMT1 expression was examined by immunocytochemistry and Northerns with an emphasis on the prenatal period. In the female, there is a gradual down-regulation of DNMT1 protein in prenatal meiotic prophase I oocytes that is not associated with the production of an untranslated transcript, as it is in the male; these results suggest that the mechanism of meiotic down-regulation differs between the sexes. In the male, DNMT1 is unlikely to play a role in the prenatal acquisition of germ line methylation patterns since it is down-regulated in gonocytes between 14.5 and 18.5 days of gestation and is absent at the time of initiation of DNA methylation. To search for candidate DNMTs that could be involved in establishing methylation patterns in both germ lines, real-time RT-PCR was used to simultaneously study the expression profiles of the three DNMT3 enzymes in developing testes and ovaries; DNMT1 expression was included as a control. Expression profiles of DNMT3a and DNMT3l provide support for an interaction of the two enzymes during prenatal germ cell development and de novo methylation in the male. DNMT3l is the predominant DNMT3 enzyme expressed at high levels in the postnatal female germ line at the time of acquisition of DNA methylation patterns. DNMT1 and DNMT3b expression levels peak concomitantly, shortly after birth in the male, consistent with a role in the maintenance of methylation patterns in proliferating spermatogonia. Together, the results provide clues to specific roles for the different DNMT family members in de novo and maintenance methylation in the developing testis and ovary.

The imprinting of mammalian genes depends on the maintenance of DNA methylation patterns during pre- and postimplantation development. Dnmt1o is a variant form of the somatically expressed Dnmt1 cytosine methyltransferase that is synthesized and stored in the oocyte cytoplasm and trafficks to the eight-cell nucleus during preimplantation development, where it maintains DNA methylation patterns on alleles of imprinted genes. Transcripts encoding Dnmt1 are present in preimplantation embryos, suggesting that Dnmt1 protein is also expressed in the preimplantation embryo, and may account for maintenance methylation at preimplantation stages other than the eight-cell embryo. However, using an antibody that detects Dnmt1, but not Dnmt1o, no Dnmt1 protein was detected on immunoblots or by immunocytochemical staining in wildtype preimplantation embryos. Moreover, Dnmt1 protein produced in the oocyte from a modified Dnmt1 allele, Dnmt1(1s/1o), trafficked to nuclei of eight-cell embryos, but not to nuclei of other stages. The highly restricted nuclear localization patterns of oocyte-derived Dnmt1o and Dnmt1 during preimplantation development add further support to the notion that DNA methyltransferases other than Dnmt1 are required for maintaining imprints during preimplantation development.

Cellular junctions in the testis and epididymis play crucial roles for the development and maturation of spermatozoa. In the testis, tight junctions between Sertoli cells form a functional blood testis barrier between 10 and 16 days of age, whereas the tight junctional blood epididymal barrier between adjacent epithelial cells is formed between days 18 and 21. In the present study, occludin, a constituent integral membrane protein of tight junctions, was localized by immunofluorescent confocal microscopy in embryonic (days 13.5-18.5), postnatal (days 5-23) and adult (day 70) mouse testes and epididymides to correlate its expression with the onset of tight junctions and eventual formation of these barriers. At embryonic days 13.5 and 16.5, low diffuse cytoplasmic levels of occludin were observed in cells of the testicular cords. By embryonic day 18.5, the level of occludin was still low but appeared as a filiform-like network streaming toward the center of the cord. At postnatal days 5 and 7 immunostaining became more intense and appeared to outline the periphery of Sertoli cells of seminiferous tubules. Postnatal day 14 marked the appearance of an intense, focal band-like localization of occludin at the base of the tubules, correlating with the appearance of a functional blood-testis barrier. By day 23 and in adults, expression of occludin was noted at the base of the tubule appearing as intense, wavy, discontinuous bands similar in appearance irrespective of the stage of the seminiferous epithelium cycle. In the developing epididymis, intense cytoplasmic immunostaining was present in epithelial cells of many epididymal tubules at embryonic day 13.5. By embryonic day 16.5, intense occludin immunostaining appeared along the lateral plasma membranes of epithelial cells, whereas at embryonic day 18.5, immunostaining was punctate and apically located, suggesting the presence of tight junctions by this age; similar immunostaining was noted at postnatal days 5 and 7. In the adult epididymis, distinct punctate apical staining was observed between adjacent principal cells of all epididymal regions except the proximal initial segment, where occludin was found only in association with narrow cells. These results indicate that in the epididymis, the appearance of occludin at apical sites between adjacent epithelial cells occurs during embryonic development suggesting that tight junctions form earlier than in the testis. While occludin was expressed in a similar pattern between Sertoli cells at all stages of the cycle in the adult testis, its expression in the adult epididymis was cell- and region-specific. Taken together these data suggest that different factors regulate occludin expression in the testis and epididymis.

Lysosomal sialidase occurs in a multienzyme complex that also contains beta-galactosidase and cathepsin A. We previously cloned the human lysosomal sialidase cDNA and characterized mutations in human sialidosis patients. Here, we report the cloning and expression of the mouse lysosomal sialidase cDNA and gene. The 1.77 kb cDNA encodes an open reading frame of 408 amino acids which shows high homology to the human lysosomal sialidase (80%), the rat cytosolic sialidase (65%) and viral and bacterial sialidases (50-55%). The sialidase gene is approximately 4 kb long and contains six exons. The five introns range in size from 96 to 1200 bp. Northern blot analysis revealed high expression of multiple sialidase transcripts in kidney and epididymis, moderate levels in brain and spinal cord, and low levels in adrenal, heart, liver, lung and spleen. Transient expression of the cDNA clone in sialidase-deficient SM/J mouse fibroblasts and human sialidosis fibroblasts restored normal levels of sialidase activities in both cell types. Immunocytochemically expressed sialidase co-localized with a lysosomal marker, LAMP2, confirming its lysosomal nature. Since sialidase activity requires its association with beta-galactosidase and cathepsin A, the expression of mouse sialidase within human sialidosis cells underlines the structural similarity between mouse and human enzymes and suggests that the mechanism for complex formation and function is highly conserved.

Mammalian gonadal development and sexual differentiation are complex processes that require the coordinated expression of a specific set of genes in a strict spatiotemporal manner. Although some of these genes have been identified, the molecular pathways, including transcription factors, that are critical for the early events of lineage commitment and sexual dimorphism, remain poorly understood. GATA-4, a member of the GATA family of transcription factors, is present in the gonads and may be a regulator of gonadal gene expression. We have analyzed the ontogeny of gonadal GATA-4 expression by immunohistochemistry. GATA-4 protein was detected as early as embryonic day 11.5 in the primitive gonads of both XX and XY mouse embryos. In both sexes, GATA-4 specifically marked the developing somatic cell lineages (Sertoli in testis and granulosa in ovary) but not primordial germ cells. Interestingly, abundant GATA-4 expression was maintained in Sertoli cells throughout embryonic development but was markedly down-regulated shortly after the histological differentiation of the ovary on embryonic day 13.5. This pattern of expression suggested that GATA-4 might be involved in early gonadal development and possibly sexual dimorphism. Consistent with this hypothesis, we found that the Mullerian inhibiting substance promoter which harbors a conserved GATA element is a downstream target for GATA-4. Thus, transcription factor GATA-4 may be a new factor in the cascade of regulators that control gonadal development and sex differentiation in mammals.

The spermatozoon and oocyte genomes bear sex-specific methylation patterns that are established during gametogenesis and are required for the allele-specific expression of imprinted genes in somatic tissues. The mRNA for Dnmt1, the predominant maintenance and de novo DNA (cytosine-5)-methyl transferase in mammals, is present at high levels in postmitotic murine germ cells but undergoes alternative splicing of sex-specific 5′ exons, which controls the production and localization of enzyme during specific stages of gametogenesis. An oocyte-specific 5′ exon is associated with the production of very large amounts of active Dnmt1 protein, which is truncated at the N terminus and sequestered in the cytoplasm during the later stages of oocyte growth, while a spermatocyte-specific 5′ exon interferes with translation and prevents production of Dnmt1 during the prolonged crossing-over stage of male meiosis. During the course of postnatal oogenesis, Dnmt1 is present at high levels in nuclei only in growing dictyate oocytes, a stage during which gynogenetic developmental potential is lost and biparental developmental potential is gained.