Investigating in utero Fertilization, in vitro Fertilization, and the Mermaid-2 Voltage Sensor in Caenorhabditis elegans Open Access

Phillis, Hugh (2017)

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C. elegans share a genetic homology with humans and thus may provide valuable insights into human biology, chemistry, and more. The L'Hernault lab identifies and investigates fertilization defective mutant strains of C. elegans. The spe-9 class mutants are of interest because they produce seemingly normal spermatozoa, but are unable to fertilize an oocyte. However, tools need to be developed to further study, evaluate, and classify these mutants. My objectives were to induce and document in utero fertilization, develop a protocol for in vitro fertilization, and create a voltage sensor to investigate ion shifts during fertilization. I have gathered a substantial amount of evidence proving in utero fertilization of nonEmo oocytes can occur. Normally, fertilization occurs in the spermatheca and no prior work has found it to occur elsewhere in C. elegans. One reason is that wild-type oocytes complete meiosis upon entering the spermatheca and undergo endomitosis (Emo) if they fail to be fertilized. Prior work has shown that the pezo-1 mutant forms oocytes that remain in meiosis, even if they are not fertilized. These nonEmo oocytes were evaluated in my work to explore if they could be fertilized outside of the spermatheca. This suggests that the mutant nonEmo oocyte phenotype maintains fertilization competency after passing through the spermatheca. In addition, it also suggests that spermatozoa do not undergo any special capacitation event within the spermatheca. Most importantly, it signifies that in vitro fertilization might be possible. However, in vitro fertilization has yet to be realized. Lastly, I have successfully created a plasmid containing the Mermaid-2 voltage sensor and have used transgenic techniques to induce its expression in the germline of C. elegans. Future research must center on utilizing the Mermaid-2 voltage sensor to evaluate fertilization defective mutants and successfully completing in vitro fertilization. These efforts may one day reveal new insights in to mammalian fertilization and be used in the treatment of infertility.

Table of Contents

Table of Contents

Chapter I: Introduction (1)

A. Overview of Caenorhabditis elegans (C. elegans) (1)

B. Hermaphroditic Reproductive Anatomy (2)

C. Male Reproductive Anatomy (2)

D. Fertilization in C. elegans (3)

Chapter II: in utero Fertilization (IUF) (7) A. Introduction (7)

B. Materials and Methods (7)

C. Results (10) D. Discussion (12) Chapter III: in vitro Fertilization (IVF) (15) A. Introduction (15)

B. Materials and Methods (17)

C. Results (22) D. Discussion (24)

Chapter IV: Creating Mermaid-2Expression in C. elegans (26)

A. Introduction (26)

B. Materials and Methods (29)

C. Results (31) D. Discussion (32)

Chapter V: Future Directions (34)

Figures and Tables (36)

References (70)

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