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.