Mechanisms of Double Strand Break Repair in Fetal Male Germ Cells

Male germ cells (MGCs) are precursors to spermatogonial stem cells (SSCs), a stem cell population that both self-renews and differentiates, supplying spermatozoa for the entirety of a male’s reproductive lifespan. Therefore, any genomic perturbation in MGCs caused by inaccurate DNA damage repair can be heritable. Just prior to birth, MGCs undergo a relatively long period of cellular quiescence (G0). This period is required for SSC development and is developmentally conserved between mice and humans. During this G0 phase, MGCs experience hyper-transcription and changes in epigenetic modifications, two cellular activities that directly cause DNA damage in somatic cells. However, little is known about the DNA damage that accumulates in MGCs during this G0 arrest or how it is repaired to maintain fidelity of the germline genome. The most potent form of DNA damage is double strand breaks (DSBs). DSBs are often repaired by one of two pathways, non-homologous end joining (NHEJ) or homologous recombination (HR). NHEJ is an efficient, yet error-prone repair mechanism that is used in the G0/G1 stages of the cell cycle. In contrast, HR, which requires a homologous template, is a more complex, yet accurate repair mechanism that is used in S and G2 stages of the cell cycle. In G0 MGCs, maintaining the integrity of the genome during DSB repair is of utmost importance. However, the mechanism in which G0 MGCs repair DSBs is unknown. The overall objective of this K99/R00 proposal is to determine how G0 MGCs respond to and molecularly repair DSBs and to elucidate the importance of G0 MGC DSB repair in SSC development. The central hypothesis, which is based on preliminary data, is that G0 MGCs employ a modified-HR mechanism that incorporates NHEJ proteins, using the homologous chromosome as a template to repair DSBs and ensuring SSC development. In Aim 1 (K99 phase), single cell transcriptomics, immunofluorescence, and genetic models will be used to define the pathway that MGCs employ to repair DSBs. In Aim 2 (K99/R00 phase), a novel, in vivo CRISPR-Cas9 based assay will be generated and used to elucidate the molecular mechanism of DSB repair. In Aim 3 (R00 phase), genetic models, single cell transcriptomics, and single cell genomics will determine the implications of DSB repair defects in G0 MGCs on SSC development. Data and skills obtained from the K99 portion of this proposal will provide the basis for a strong and innovative independent research program, which will be expanded upon using data obtained from the R00 phase. Completion of these Aims will require training in complex bioinformatic analyses and mouse genetics, and the enclosed development plan describes a two-year blueprint designed to strengthen these skills. The research and development plans will be implemented at Duke University in the laboratory of Dr. Blanche Capel. Overall, the enclosed K99/R00 proposal can successfully transition a mentored scientist into a creative and skilled independent researcher and significantly advance knowledge for the improvement of reproductive health. Project Number: 1K99HD117011-01 | Fiscal Year: 2025 | NIH Institute/Center: Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) | Principal Investigator: Talia Hatkevich | Institution: DUKE UNIVERSITY, DURHAM, NC | Award Amount: $107,458 | Activity Code: K99 | Study Section: Reproduction, Andrology, and Gynecology Study Section[CHHD-R] View on NIH RePORTER: https://reporter.nih.gov/project-details/1K99HD11701101