Sexual Differentiation and Primordial Germ Cell Distribution in the Early Horse Fetus

SIMPLE SUMMARY: In horses, gonadal development and sexual differentiation occur during early fetal life. This is accompanied by primordial germ cell differentiation and migration to the gonad site. However, little is known about the time when these processes take place and the mechanisms behind them. Additionally, no information is available regarding number and distribution of primordial germ cells in the equine gonad. During development and differentiation, gonads can be affected by stressors causing infertility, but this condition can only be diagnosed in post-pubertal animals. Herein we show that equine gonads develop asynchronously between male and female fetuses, and that the number and distribution of primordial germ cells is influenced by fetal sex. By day 45 of pregnancy, equine gonads were clearly differentiated, while migration of primordial germ cells still occurred at this stage. No sign of genital duct regression was seen until day 60 of pregnancy. ABSTRACT: It was the aim of this study to characterize the development of the gonads and genital ducts in the equine fetus around the time of sexual differentiation. This included the identification and localization of the primordial germ cell population. Equine fetuses between 45 and 60 days of gestation were evaluated using a combination of micro-computed tomography scanning, immunohistochemistry, and multiplex immunofluorescence. Fetal gonads increased in size 23-fold from 45 to 60 days of gestation, and an even greater increase was observed in the metanephros volume. Signs of mesonephros atrophy were detected during this time. Tubular structures of the fetal testes were present from day 50 onwards, whereas cell clusters dominated in the fetal ovary. The genital ducts were well-differentiated and presented a lumen in all samples. No sign of mesonephric or paramesonephric duct degeneration was detected. Expression of AMH was strong in the fetal testes but absent in ovaries. Irrespective of sex, primordial germ cells selectively expressed LIN28. Migration of primordial germ cells from the mesonephros to the gonad was detected at 45 days, but not at 60 days of development. Their number and distribution within the gonad were influenced (p < 0.05) by fetal sex. Most primordial germ cells (86.8 ± 3.2% in females and 84.6 ± 4.7% in males) were characterized as pluripotent according to co-localization with CD117. However, only a very small percentage of primordial germ cells were proliferating (7.5 ± 1.7% in females and 3.2 ± 1.2% in males) based on co-localization with Ki67. It can be concluded that gonadal sexual differentiation in the horse occurs asynchronously with regard to sex but already before 45 days of gestation.