Knockout of Highly Conserved Seminal Fluid Protein Ablates Male Fertility in Drosophila melanogaster
Date of Award
5-12-2024
Degree Type
Thesis
Degree Name
Master of Science (MS)
Department
Biology
Advisor(s)
Yair Ahmed-Braimah
Keywords
Drosophila;Glycosylation;Reproduction;β-Glucuronidase
Subject Categories
Biochemistry, Biophysics, and Structural Biology | Life Sciences | Molecular Biology
Abstract
ABSTRACT In Drosophila, seminal fluid proteins (SFPs) are key components of the male ejaculate and are essential determiners of reproductive fitness. SFPs are required for inducing a range of post-mating physiological responses in females. To date nearly 300 SFPs have been identified, however the vast majority of these remain uncharacterized and their role in reproduction and/or postcopulatory sexual selection is not clear. SFPs are functionally diverse and contain proteins from a variety of biochemical processes, most notably proteases and protease inhibitors that are thought to be critically important in biochemical interactions with female proteins. Another class of proteins that is enriched among SFPs are carbohydrate bonding proteins, presumably involved in glycolytic reactions within the accessory glands and in mated females. However, the molecular mechanisms and fertility roles of these ejaculate glycolysis modulators have yet to be explored. One of these uncharacterized glycolysis SFPs is the highly conserved beta-glucoronidase (CG15117), which catalyzes the breakdown of complex carbohydrates. CG15117—which I will refer to as BGLUC—is (1) predominately expressed in the male accessory gland—but maintains low level expression in other tissues, (2) is transferred to the female during mating, and (3) is one of the most conserved SFPs in Drosophila. To identify BGLUC’s role in reproduction, we created a CRISPR/Cas-9 knockout mutant by ablating 5 bp from the coding sequence and introducing a premature stop codon. We found that BGLUC is required for male fertility: females that are mated to knockout males do not produce progeny, fail to store sperm and will readily remate, suggesting that an ensemble of key post-maing processes is disrupted. To further examine the molecular basis of this male sterility we performed label-free quantitative proteomic analysis and bulk RNA sequencing on mutant males as well as females mated to mutant and control males and find systematic abnormal abundance of several proteins—including proteins of ACP36DE and ACP62F— in the accessory glands as well as ACP36DE and other proteins in the transferred male ejaculate. ACP36DE and ACP62F have major roles in modulating sperm storage in the female and their reduced abundances in mutant male accessory glands likely contributes to sperm storage defects observed in females mated by mutant males. Together, our results show that the often-ignored carbohydrate metabolism proteins that are part of the seminal plasma are essential for fertility in Drosophila.
Access
Open Access
Recommended Citation
Hockenbury, Dominic, "Knockout of Highly Conserved Seminal Fluid Protein Ablates Male Fertility in Drosophila melanogaster" (2024). Theses - ALL. 890.
https://surface.syr.edu/thesis/890
