Investigating How DNA Organization Contributes to Spermatogenesis and Zygotic Development

Date of Award

Fall 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Genetics

First Advisor

Lesch, Bluma

Abstract

Although male infertility is a global issue, little is known regarding its molecular causes. Currently, patients are assessed based on physical measurements, such as sperm morphology, motility, and concentration. Assisted reproductive technologies, such as intracytoplasmic sperm injection (ICSI), were created to artificially fertilize oocytes by directly injecting a sperm head, bypassing problems such as sperm motility. While this procedure circumvents any physical means of infertility, it does not address underlying molecular causes that might arise from improper gamete differentiation. Here, I investigate how DNA organization impacts spermatogenesis and embryogenesis through multiple gene knock-out (KO) mouse models. To show how protamine 2 (Prm2) deficiency changes zygotic development, sperm from a mouse with Prm2 deficiency was injected into a control oocyte. The resulting zygote arrested at the 2-cell stage after a fragmented first cleavage division, likely due to the dissipation of the paternal DNA within 2 hours post-fertilization, and the formation of an abnormal maternal pronucleus. These phenotypes were not recapitulated using artificially DNA damaged sperm, suggesting damage alone is not responsible for these phenotypes. Co-injection with a Prm2 KO sperm and a fully-protaminated paternal genome could not rescue these phenotypes. However, ICSI using testicular sperm from Prm2 KO mice resulted in developed blastocysts, suggesting these deleterious factors are gained during epididymal transit. These data show how changing DNA packaging in a sperm can influence the development of an embryo. To further characterize how chromatin can impact spermatogenesis and fertility, I investigated the transcriptional states of cells during spermatogenesis when histone modifiers KDM6A and DOT1L are removed, along with the chromatin remodeler SMARCA5 and packaging protein PRM2, through single cell RNA-sequencing. Each phenotype is unique to its corresponding deficiency, supporting the idea that chromatin remodeling plays multiple roles in spermatogenesis and resulting zygotes. In summary, the packaging of the genome during spermatogenesis plays a significant role in spermatogenesis and the fertilization capacity of epididymal sperm.

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