46 Morphologic and functional characterization of the early fetal equine gonads

Abstract

In the equine embryo, putative primordial germ cells appear between 20 and 30 days and the gonadal primordium can first be identified at Day 30 after ovulation, respectively. Subsequently, sexual differentiation of the gonad occurs and completes by Day 45 of pregnancy. The objectives of this work were to describe the morphology and function of the fetal equine ovary and testis at the beginning of the fetal stage of pregnancy. For this purpose, 12 equine fetuses (6 males and 6 females) were collected at 45 days (n = 1, female), 50 days (n = 1, male), and 60 days (n = 10, 5 males and 5 females) after ovulation, respectively. A high attention was given to Day 60 because it is the representative time for fetal sex determination in horses by transrectal ultrasonography. Conceptuses were collected transcervically by uterine lavage and fixed in 4% formaldehyde before being prepared for morphology analysis and immunohistochemistry assay. Gonads were identified and immunostained for anti-Müllerian hormone (AMH), Ki67, CD117, LIN28, vimentin, cytokeratin, and laminin. In all fetuses, gonads were situated in a sublumbar localisation and connected with the mesonephros. In females, primordial germ cells were localised close to the surface germinal epithelium, whereas in males the primordial germ cells were organised in cord-like clusters-the future seminiferous tubules. At this stage, interstitial cells predominate in the testes. The AMH staining was strongly expressed in the fetal testis, but was completely absent from the fetal ovary. Protein expression of mitosis marker Ki67 was localised in primordial germ cells of both sexes. Moreover, stem cell markers LIN28 and CD117 were also present in the gonads. In females, these proteins were not only localised in some of the primordial germ cells, but also in the surface germinal epithelium, whereas in males LIN28 and CD117 were immunolocalized in the seminiferous tubules, distant from the surface epithelium. Vimentin was strongly expressed in the interstitial cells of the gonads of both sexes. Using laminin staining, basal membrane of the seminiferous tubules in males and of primordial germ cells in females could be visualised. In females, the basal membrane of primordial germ cells also stained positive for cytokeratin, whereas in males no cytokeratin staining was seen around seminiferous tubules. Moreover, the surface germinal epithelium of both sexes stained positive for cytokeratin. This study widely extends existing knowledge about morphology, development, and function of the early fetal equine gonad. Presence of stem cells could be clearly demonstrated in the gonads of both sexes, whereas AMH staining clearly distinguished between males and females, confirming the important role of this hormone for gonadal and reproductive tract differentiation.