Identifying disruptors of male germ cell development by small molecule screening in ex vivo gonad cultures

Abstract

BackgroundGerm cell development involves formation of the spermatogenic or oogenic lineages from the bipotential primordial germ cells. Signaling mechanisms in the fetal testis and ovary determine whether germ cells enter the male or female developmental pathway, respectively. These signaling processes underpin an important phase of germ cell development, disruption of which can lead to failed germ cell function resulting in infertility or the formation of germ cell tumours.FindingsIn this study we have developed a small molecule screening protocol combined with flow cytometry to identify signaling pathways that direct male-specific development of germ cells. Here we provide a detailed method for this screening protocol, which we have used to identify signaling pathways important for male germ cell development.ConclusionThis method will be of particular use in screening inhibitors of signaling pathways, endocrine disruptors or other chemicals for their ability to disrupt testis and germ cell development, thereby providing insight into testicular dysgenesis and factors underlying poor male reproductive health.

The Aorta-Gonad-Mesonephros Organ Culture Recapitulates 5hmC Reorganization and Replication-Dependent and Independent Loss of DNA Methylation in the Germline

“…5A, B). In this assay, the cell cycle dynamics of PGCs were distinguished from somatic cells in the same sample using intracellular staining for Mvh [28]. Similar to previous reports [19], our data show that the somatic cells of the organ culture are mostly in the G1 phase of the cell cycle (73%-75%).…”

“…Further analysis and quantification were performed using FlowJo (version 9.3.13). For detailed information of how to set up the gates for cell cycle analysis, refer to Wakeling et al [28].…”

“…Staining of the harvested AGMs was performed according to previous published studies with intracellular staining for Mvh detected with Alexa Flour 488 used to discriminate germline cells from nongerm cells [28]. Samples were analyzed using the LSRII (BD) flow cytometry machine.…”

Global Hypertranscription in the Mouse Embryonic Germline

“…Nascent RNA was labeled with Click-iT RNA imaging kits (Thermo Fisher Scientific), followed by incubation with anti-MVH (Abcam, ab13840) to stain PGCs and GCs, as described previously (Wakeling et al, 2013). …”

A detailed protocol for a rapid analysis of testicular cell populations using flow cytometry

“…An obvious limitation of intracellular flow cytometry is that it does not provide viable sorted cells for downstream analyses as the cell are fixed and permeabilized during the sample preparation. In the context of testicular research, intracellular flow cytometry has been previously used to study proliferating cell populations in the fetal and adult mouse testes (Elzeinova et al ., 2013; Ferguson et al ., 2013; Wakeling et al ., 2013). …”