Title

Sperm Proteome Variation between Species and within the Female Reproductive Tract

Date of Award

December 2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Advisor(s)

Steve Dorus

Keywords

proteomics, sexual selection, sperm

Subject Categories

Life Sciences

Abstract

Sperm are under the contrasting selective forces of strong purifying selection, associated with their critical function in reproduction, and intense postcopulatory sexual selection, in the form of cryptic female choice and sperm competition. As a consequence of postcopulatory sexual selection, sperm are among the most rapidly diversifying cell types known and exhibit considerable variation in morphology, physiology, biochemistry, and behaviour. The more recent application of mass spectrometry-based proteomic techniques to sperm research has revealed this diversity extends into the molecular level. One example of this diversity is sperm heteromorphism. The phenomenon in which a male produces multiple discrete sperm morphs in a single ejaculate, only one of which is fertilisation competent. Sperm heteromorphism is nearly universal in Lepidoptera, with males producing a fertilisation competent sperm morph (“eupyrene”) containing the full haploid genetic complement and an incompetent sperm morph (“apyrene”) that lacks nuclear DNA entirely. Here we characterised the proteome of both sperm morphs in the Carolina sphinx moth (Manduca sexta) and the monarch butterfly (Danaus plexippus). We found an enrichment for Lepidoptera novel genes encoding sperm proteins and reduced orthology in non-Lepidopteran insect taxa consistent with a burst of novelty concurrent with the origin of sperm heteromorphism in this group. Our comparative analyses of apyrene and eupyrene sperm revealed that proteins common to both morphs were more conserved as sperm components, whereas morph-specific proteins turned over more quickly. In particular, the apyrene sperm proteome was found to diverge more rapidly and be less complex with evidence of lineage specific gene loss. This may be consistent with a relaxation of selective constraints associated with fertilisation or adaptive responses to sexual selection. Sperm are primarily characterised using samples obtained from the male, however sperm diversity does not end at ejaculation. Post-Ejaculatory Modifications to Sperm (PEMS) introduce a post-copulatory level to sperm diversity, are observed in a taxonomically broad range of species, are likely critical to fertilisation, and may play a role in postcopulatory sexual selection and reproductive isolation. Despite their apparent prevalence and importance, current observations of PEMS are descriptive in nature and based on postmating differences in sperm ultrastructure and behaviour. Here, we applied tandem mass spectrometry-based proteomic methods to characterise changes in the D. melanogaster sperm proteome at four biologically relevant postmating collection points in the FRT. This was coupled with isotopic labelling techniques to confidently identify female-derived proteins associating with sperm. We identified 183 SFPs as transferred to the female during copulation, 96 of which accompanied sperm into the storage organs. Sex-specific isotopic labelling identified a total of 461 female-derived proteins, 60% (277 of 461) of which have previously been identified as components of sperm in the male seminal vesicle. These female-derived sperm components were enriched for proteins functioning in metabolism and energetics, proposing that they may maintain sperm metabolism and survival during storage. The remaining female-derived proteins identified were enriched for the proteasome complex and TCP-1 domain containing chaperonins. This suggested that females may mediate the degradation of dead or damaged sperm and remodel protein structure during storage. Our characterisation of the Drosophila post-insemination sperm proteome provides a list of candidates for interacting proteins underlying PEMS for future study.

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