Suitability of an In Vitro Cell-Based Meiosis Initiation Assay for High-Throughput Drug Screening




Goyins, Krystal

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Non-hormonal contraceptive drug delivery to meiotic and post-meiotic germ cells is often impeded by the blood-testis barrier; however, spermatogonia and early spermatocytes are present on the basal side of the barrier and are far more susceptible to drug targeting. Therefore, the purpose of my thesis was to miniaturize an in vitro meiotic initiation assay of synchronized male germ cells from 96 well plates, the current format, to 384 well plates, and automate the process with a liquid handler with the intent of conducting a high-throughput drug screen to identify compounds which inhibit meiotic entry. Neonatal male mice were treated to synchronize spermatogenesis and used to prepare primary testis cell cultures maintained concurrently in 96 and 384 well plates. Germ cells were evaluated for meiotic (SYCP3) and premeiotic (DMRT1) markers to determine meiotic entry and progression during the culture period. Testis sections obtained from similarly treated mice were evaluated for SYCP3 and DMRT1 to determine if germ cells in vitro recapitulated meiotic entry and progression observed in vivo over the same time points. Results from these experiments revealed inconsistencies of DMRT1 germ cell labeling within in vivo replicates and SYCP3 labeling in premeiotic germ cells in vitro. The use of an automated liquid handler was determined to be infeasible for this assay due to marked germ cell loss observed in both well plate formats. Further optimization of the current in vitro meiotic initiation assay is required to conduct and draw reliable conclusions from a high-throughput drug screen.


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Suitability, In vitro cell, Meiosis, Drug screening



Neuroscience, Developmental and Regenerative Biology