57 Primordial germ cell distribution in the horse fetal gonad

Abstract

Germ cell development and differentiation is a complex process associated with down-regulation of stem cell-associated genes and up-regulation of markers of germ cell differentiation and meiosis. In horses, putative primordial germ cells (PGCs) were identified outside the gonads starting 20 days after ovulation (Curran et al. 1997 Equine Vet. J. Suppl. 25, 72-76). However, no information is available after the time when these cells enter the gonad. The aim of this study was to identify, localise, and quantify PGCs in fetal male and female gonads. Twelve (5 males and 7 females) equine fetuses were collected transcervically 60 days after ovulation. Fetuses were entirely fixed in formaldehyde and gonads were subsequently localised. Fetal gonads underwent multiplex immunofluorescence staining for pre-meiotic germ cell marker LIN28, stem-cell associated marker CD117, and cell proliferation marker Ki67. Specificity of the primary antibodies for equine samples has been first validated. Quantification of fluorescence positive areas for LIN28, CD117, and Ki67 in the fetal gonads was done using a macro for ImageJ. Furthermore, we analysed the co-localization of above-mentioned markers using the same macro. Statistical comparison for differences between males and females was performed using non-parametric tests. In both sexes, PGCs proliferated as determined by double immunofluorescence of Ki67 and LIN28. Protein expression of LIN28 and Ki67 was highly correlated (r = 0.92; P = 0.003). In the fetal female gonad, PGCs were organised in cord-like structures localised in the cortical region, but there were also LIN28+ cells in the surface germinal epithelium. In the fetal male gonad, PGCs were restricted to the already developed tubular structures. Fewer LIN28+ cells (3.0 ± 0.4% vs. 4.5 ± 0.3%; P < 0.05) were present in female than in male gonads. The distribution pattern of the stem cell factor receptor CD117 was similar to LIN28, as 86.8 ± 3.2% of LIN28+ cells in females and 84.6 ± 4.7% in males were also CD117+. However, immunofluorescent co-localization analysis revealed a subpopulation of CD117+ cells (43.1 ± 8.1% in females and 46.1 ± 6.1% in males), which did not show an overlap with LIN28. These were presumably stem cells localised in the medullar area of the gonad. In summary, we analysed for the first time spatial distribution of PGCs in fetal equine gonads. We demonstrated LIN28 to be a specific marker for PGCs also in the horse gonad, which is in agreement with the situation in human and other species. Furthermore, we identified stem cells and described their localization in the fetal equine gonad. Nevertheless, the temporal distribution of PGC and stem cells in the developing horse gonad and the role of LIN28 in the maintenance of the germline stem cell state still need to be investigated.