Use of a Heterologous Promoter, mes-4, and met-2 to Study spe-5 function during C. elegans Spermatogenesis Open Access

Kaila, Vishal R (2012)

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Use of a Heterologous Promoter, mes-4, and met-2 to Study spe-5 function during C. elegans Spermatogenesis

By Vishal Reddy Kaila

Fusion of secretory vesicles with the cell membrane creates a spermatozoan surface needed for successful fertilization in the nematode Caenorhabditis elegans. Acidification of sperm-specific secretory vesicles, named fibrous body-membranous organelles (FB-MOs), is vital for proper fusion with the cell membrane. This acidification employs the vacuolar H+ ATPase (V-ATPase) that functions across the membrane of secretory vesicles and we hypothesize that spe-5, a gene that encodes a B subunit of the V-ATPase, participates in this process. There are two known paralogs that encode a B subunit of the V-ATPase in C. elegans: spe-5 and vha-12. Both genes have similar amino acid sequences (~83%) but have different chromosomal locations. spe-5 is found on chromosome I and vha-12 is on the X chromosome, which is silenced during spermatogenesis. Mutants in spe-5 are sterile and produce no progeny. However, an extrachromosomal array with vha-12 driven by its own promoter in a spe-5 mutant displayed a partial rescue of the self-sterile phenotype of this mutant. In this study, C. elegans spe-5 mutants were crossed into met-2, a gene that blocks the germline specific X chromosome silencing seen in C. elegans mutants, however the resulting double mutant yielded no progeny. In addition, met-2 and mes-4, another gene that blocks germline specific X chromosome silencing in C. elegans, were targeted via RNAi in spe-5 mutants. While spe-5 mutants subjected to met-2 RNAi did not have increased progeny, spe-5 mutants that had RNAi knockdown of mes-4 had an increase in the number of progeny. Morphology of sperm in spe-5 mutants subject to mes-4 RNAi was affected so that they became similar to wild type sperm. These data suggest that misexpression of vha-12 in its native location on the X-chromosome can partially compensate for thedefects seen in spe-5 mutants, presumably by restoring function during C. elegans spermatogenesis. In future experiments, spe-5 mutants containing a copy of spe-5, driven by the vha-12 promoter will be examined to determine transcriptional regulation of spe-5 during spermatogenesis.

Table of Contents

Table of Contents Chapter I. Introduction 1 A. Overview of Caenorhabditis elegans 2 B. Spermatogenesis 3 C. The Fibrous Body-Membranous Organelle 4 D. Mutations affecting spermatogenesis 5 Chapter II. Literature Review of Vacuolar ATPase 9 A. Overview of V-ATPase 10 B. Structure and function of V-ATPase 11 C. Regulation of V-ATPase activity 14 D. V-ATPase and vesicular trafficking 16 Chapter III. Germ-line silencing of the X chromosome in C. elegans 18 19 A. Overview 19 B. X- chromosome silencing in the germ line 20 C. XO male heterochromatin dynamics 20 D. MES histone modifiers 23 Chapter IV. Factors that Control V-ATPase B subunit Expression during C. elegans Spermatogenesis A. Introduction to SPE-5 24 B. Materials and methods 25 C. Results 30 D. Discussion 35 E. Future directions 36 References 37 Tables and Figures 48

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