Distribution of putative primordial germ cells in equine embryos.

Eighteen equine embryos, 3 each on Days 20, 22, 24, 26, 28 and 30 post ovulation, were collected transcervically by uterine lavage, fixed in 4% paraformaldehyde and embedded in paraffin wax. Ten micron serial sections were stained to determine alkaline phosphatase (AP) activity in the cells. Positive cells were counted and their approximate location determined. The cells were approximately 8 microm in diameter and the entire cell, except the nucleus, stained strongly with many small round areas of intense staining in the cytoplasm. The cells varied from round to elongated in shape and pseudopodia were often present. Thus, they were similar in shape and staining pattern to primordial germ cells described in other species. A total of 2 AP-positive cells were found in the 3 Day 20 embryos and the mean number of AP-positive cells changed (P<0.05) over the succeeding days as follows; Day 20 = 1; Day 22 = 251; Day 24 = 1484; Day 26 = 2385; Day 28 = 3267; Day 30 = 2424. AP-positive cells present in the liver were not included in the calculations. In the Day 22 embryos, 10% of the putative primordial germ cells were found within the vascular system, including the heart chambers, and only 4% were found along the genital ridge. The percentage of cells found in the vascular system decreased from 10% on Day 22 to 1% on Day 30, although not significantly. The percentage of cells found along the genital ridge changed (P<0.05) over gestational age as follows; Day 22 = 4%; Day 24 = 10%; Day 26 = 28%; Day 28 = 28%; Day 30 = 16%. Once the putative primordial germ cells reached the developing gonads they were no longer AP-positive and nor was the gonadal stroma. The rest of the cells were distributed along the dorsal mesentery (range, 14-24%), near the dorsal aorta (16-29%), in the mesonephros (1-3%) and in other areas of the embryo (27-44%). Large numbers were in the cranial portion of the embryo. Although it is likely that the population of AP-positive cells counted included the primordial germ cells, other cells, such as haematopoietic precursor cells, could not be ruled out. The AP reactivity, the appearance of the cells and their migratory pattern through the dorsal mesentery to the gonadal ridge were consistent with descriptions of primordial germ cells in other species. Their distribution throughout the embryo, especially its cranial aspect and their location within, or in close proximity to, blood vessels suggested that the equine embryo is unusual among mammals in that some of its primordial germ cells migrate through the blood.

Differential gene expression in fetal mouse germ cells.

We have constructed cDNA libraries representing transcripts from purified populations of germ cells and of somatic cells, isolated from fetal mouse gonads at 12.5 days postcoitum (dpc). We have used these libraries to study differential gene expression in fetal germ cells on the basis of differential representation of specific cDNAs in each library. We show that in addition to expression of housekeeping genes at expected levels, four genes responsible for germ cell-specific phenotypes are expressed at significantly different levels in germ cells and surrounding somatic cells. These include tissue-nonspecific alkaline phosphatase (tnAP), transcription factor Oct-3/4, the c-kit proto-oncogene, and DNA methyltransferase (Mt). The significance of these results is discussed in the context of events contributing to early development of germ cells. It is concluded that fetal germ cells appear to retain a pattern of gene expression resembling that in early pluripotent embryonic cells, and that this may be important for the maintenance of genetic totipotency.

Developmental pattern of gene-specific DNA methylation in the mouse embryo and germ line.

Methylation patterns of specific genes have been studied by polymerase chain reaction and found to undergo dynamic changes in the germ line and early embryo. Some CpG sites are methylated in sperm DNA and unmodified in mature oocytes, indicating that the parental genomes have differential methylation profiles. These differences, however, are erased by a series of early embryonic demethylation and postblastula remodification events, which serve to reestablish the basic adult methylation pattern prior to organogenesis. During gametogenesis, all of these sites are unmethylated in primordial germ cells but eventually become remodified by 18.5 days postcoitum in both males and females. The final methylation profile of the mature germ cells is then formed by a multistep process of site-specific demethylation events. These results form a basis for the understanding of the biochemical mechanisms and role of DNA methylation in embryonic development.

Distribution of extracellular matrix in the migratory pathway of avian primordial germ cells.

The appearance and distribution of extracellular matrix (ECM) was documented along the migratory route of chicken primordial germ cells (PGCs). The antimouse embryonal carcinoma cell antibody, EMA-1, was used to label PGCs (Urven et al.: Development 103:299-304, 1988). Antibodies against laminin, fibronectin, chondroitin sulfate proteoglycan and collagen type IV were used to label extracellular matrix components. When the PGCs emerged from the epiblast, all four ECM molecules were restricted principally to the basement membrane of the epiblast. Chondroitin sulfate was also located between hypoblast cells during this period. In late germinal crescent stages, when the PGCs entered the lumina of blood vessels, the same ECM molecules were more widespread in the mesoderm and in extracellular spaces. In addition, laminin and collagen type IV were identified on lateral surfaces of ectodermal cells at this stage. When the germ cells moved through the mesenchyme into the germinal ridge, the ECM molecules were found around mesenchymal cells, and, in the cases of laminin, fibronectin and collagen type IV, in the basement membranes of the germinal ridge epithelia. Because the appearance of these ECM components is temporally and spatially correlated with the movement of PGCs, we suggest that early PGC migration may depend on their timely appearance.

Analysis of germ line development in the chick embryo using an anti-mouse EC cell antibody.

We have found that EMA-1, a monoclonal antibody originally raised against mouse embryonal carcinoma (Nulli SCC1) cells (Hahnel & Eddy, 1982), also labels chick primordial germ cells (PGCs). We have used this antibody in immunohistological studies to follow the development of PGCs in the chick embryo from the time of their initial appearance beneath the epiblast, through their migratory phase and subsequent colonization of the germinal epithelium. During hypoblast formation, individual EMA-1-labelled cells appeared to separate from the basal surface of the epiblast and enter the blastocoel, coincident with the appearance of morphologically identifiable PGCs in this same area. EMA-1 continued to label germ cells until the initiation of gametogenesis in each sex; specifically, labelling was absent by 7-8 days of incubation in females and started to decrease at 11 days of incubation in males. There was a recurrence of the epitope on oogonia at 15 days of incubation, but not on spermatogonia during the remainder of development through hatching. These observations are consistent with an epiblast origin for the avian germ line, and are strikingly similar to those reported for the early mouse embryo using the same antibody (Hahnel & Eddy, 1986).