All ETDs from UAB

Advisory Committee Chair

Michael A Miller

Advisory Committee Members

Bradley K Yoder

Jianbo Wang

Jim Collawn

Kent Keyser

Document Type

Dissertation

Date of Award

2015

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Survival of animal species depends on fertilization, the union of an egg and a sperm. The sperm's ability to find an egg quickly allows it to pass on male genetic material. It is challenging to record sperm targeting or guidance efficiency and motility in utero. We use the Caenorhabditis elegans model organism to study sperm guidance, primarily because its epidermis is transparent, allowing the observation of live fluorescent sperm in the hermaphrodite uterus. Using genome-editing techniques, genetic analyses, fluorescent microscopy, and mass spectrometry, we aim to address the following two questions: how hermaphrodites regulate sperm motility in the uterus, and how males respond to hermaphrodite cues. Chapter one gives a general introduction about fertilization, factors affecting fertilization, and sperm motility. Next, background on sperm guidance in externally and internally fertilizing species is given. Given the difficulty of observing sperm in utero in mammals, Caenorhabditis elegans provides a powerful model system to study sperm guidance. Recent findings suggest that prostaglandin-like lipids derived from C. elegans oocytes may be sperm chemoattractants. An introduction to prostaglandins, their diverse roles, their synthesis, and receptors is provided. Then background is presented on the potential impact of environment on fertility and sperm guidance. Finally, the C. elegans sensory nervous system, which is used to sense environmental cues, is discussed. In chapter two, we use genetics, liquid chromatography, and electrospray ionization tandem mass spectrometry (LC-MS/MS) to investigate prostaglandin metabolism in C. elegans hermaphrodites. Our results show that C. elegans oocytes or their precursors produce more than ten structurally related F-series prostaglandins (PGFs), which function redundantly to promote sperm targeting. PGFs are derived from omega-3 and omega-6 polyunsaturated fatty acids (PUFAs). Specifically, PGF1 stereoisomers are derived from dihomo-gamma-linolenic acid (DGLA), PGF2 stereoisomers are derived from arachidonic acid (AA) and omega-3-arachidonic acid (O3AA), and PGF3 stereoisomers are derived from eicosapentaenoic (EPA) acid. Genetic ablation of omega-3 PUFA synthesis causes a compensatory increase in production of PGFs derived from omega-6 PUFAs. C. elegans PGF synthesis occurs independent of cyclooxygenase (COX) enzymes, which initiate PGF synthesis in mammals. COX-independent PGFs containing different stereochemical configurations than PGF2α are found in mouse tissues. Finally, we identify an evolutionarily conserved cytochrome P450 enzyme that negatively regulates PGF synthesis. In chapter three, we use genome-editing and transgenic techniques, fluorescence microscopy, quantitative RT-PCR, and RNAseq to investigate the role of a G-protein coupled receptor (GPCR) subfamily in sperm guidance. We show that at least three candidate chemoreceptor GPCRs called SRB-13, SRB-16, and SRB-5 are essential in males to promote efficient sperm guidance. These SRB subfamily chemoreceptors act in at least two parallel pathways. The GOA-1 Gαi/o protein appears to be a common downstream component required for sperm guidance. srb mutant sperm are activated and motile, but do not efficiently target the fertilization site and thus, appear of poor quality. Surprisingly, SRB chemoreceptors are not expressed in sperm. Instead, they (e.g. SRB-13 and SRB-16) are expressed in male head neurons, including amphid sensory neurons that perceive environmental cues. SRB-13 is specifically expressed in the cilia of ASI and ASK amphid sensory neurons, whereas SRB-16 is expressed in cell bodies and dendrites of ASH, ASI, ASK, and AWB amphid sensory neurons. SRB-16 is also expressed in I1 pharyngeal interneurons, pm4 and pm5 pharyngeal muscles, and other unidentified male neurons. Expressing srb-16 in neurons rescues the srb-16 null mutant sperm guidance defect. Similarly, expressing srb-13 in sensory neurons rescues the srb-13 null mutant defect. RNAseq studies suggest that SRB-13 and SRB-16 regulate spermatogenic gene transcription, possibly by modulating a neuroendocrine pathway(s). Together, these data support the unexpected model that male SRB chemoreceptors couple environmental information to sperm quality. Chapter four ends the thesis by discussing additional published and unpublished data, as well as discussing remaining outstanding questions. Experimental evidence is provided that COX knockout mice synthesize PGFs. These data were published in a collaborative study with Dr. Katherine McKnight. Unpublished data document efforts to identify enzymes essential for PGF synthesis. The thesis ends with a model for the mechanisms controlling sperm guidance (a.k.a. chemotaxis) and concluding remarks on the power of the C. elegans model for generating endless exciting questions.

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