Sperm function is pivotal to successful sexual reproduction. The phenotype of sperm is defined by the male’s genotype and by the environment sperm encounter during their travel to the oocyte. During their functional lifespan, sperm encounter a variety of environments: After manufacture in the testis, they are stored in males before they are ejaculated along with seminal fluids and transferred to and stored in females for hours up to years before getting a chance to fertilise an egg. The sperm environment in male and female reproductive tract will be determined by male and female genotype, but also by environmental factors that affect sperm directly or indirectly by altering male and female condition. Like somatic cells, sperm age and decline in function over time due to the accumulation of cellular damage. Reactive oxygen species (ROS) that emerge as a
by-product of aerobic metabolism and environmental stress are believed to be the main cause of cell senescence. Spermatozoa are in particular susceptible to ROSinduced
damage because they have only limited defence and repair mechanism. As it contains many polyunsaturated fatty acids, the sperm membrane is especially prone to peroxidation by ROS and can consequently become leaky. The condition of the sperm membrane can hence be used to assess sperm age. Sperm quality has frequently been measured as sperm viability even though this approach has several biological and technical pitfalls. I developed an osmotic sperm stress test to assess sperm quality and predict future sperm performance that circumvents several of these pitfalls. Further, using osmotic stress to challenge the sperm membrane and observing sperm viability in a longitudinal approach is probably more meaningful in predicting future sperm performance than sperm viability per se. An essential abiotic factor that affects sperm directly and indirectly during storage in males and females is temperature. Ectotherms that inhabit different climates like D. melanogaster can be expected to be locally adapted to temperatures, particularly in fitness-relevant reproductive traits. I assessed the joint and isolated effects of thermal adaptation, of rearing and of ambient temperature on sperm quality by measuring sperm osmotic stress resistance, ejaculate effects on the induction of egg-laying in females, male fertility as well as female fertility and sperm storage in
two D. melanogaster strains from Zambia (warm-adapted) and two from Sweden (cold-adapted). I found complex G x E interactions on male and female reproductive traits. Sperm quality was generally higher in the cold-adapted strains and showed negative carry-over effects of hot-rearing, demonstrating the important role of male genotype and developmental temperature on sperm quality. In contrast, there were positive carry-over effects of hot-rearing on male fecundity and male and female fertilisation rate in the hot-adapted strains, supporting local adaptation to heat stress. To investigate direct effects of the female reproductive tract environment on stored sperm, I genetically manipulated female D. melanogaster with a spermathecalspecific GAL4 driver line and hoped to test proposed candidate genes associated with female sperm storage. My results suggested sperm storage defects in the driver line that may either be an unexpected side-effect of the insertion of the GAL4 driver into a spermathecal serine endopeptidase or of the genetic background of the driver line.:Chapter 1
General introduction 1
Sperm phenotype 1
Basic sperm morphology 2
Sperm ageing 3
Environmental effects on sperm 5
Male sperm storage 6
Female sperm storage (FSS) 8
Phase 1: Recruitment of sperm into storage 10
Phase 2: Sperm maintenance 11
Phase 3: Release of sperm from storage 16
References 18
Chapter 2
More pitfalls with sperm viability staining and a viability-based stress test to characterize sperm quality 29
Abstract 29
Author contributions 30
Introduction 31
Methods 33
Methods of sperm viability staining in ecology and evolution 33
Empirical study 33
Statistical analysis 38
Results 38
Methods of sperm viability staining in ecology and evolution 38
Sperm survival examined with cross-sectional vs. longitudinal sampling 43
Sperm viability in the bedbug 43
Sperm viability in the fruitfly 45
Discussion 47
SV heterogeneity 48
Protocol standardization and recommendations 49
Sperm viability vs. sperm quality 50
Sperm stratification 51
Conclusion 52
References 52
Chapter 3
Effects of temperature and thermal adaptation on sperm stored in male and female Drosophila melanogaster 56
Abstract 56
Introduction 57
Material and Methods 61
Fly populations and culture 61
Temperature treatments 62
Wing length 63
Quality of sperm stored in males 63
Male effects on female fertility 64
Female effects on sperm 64
Statistical analysis 66
Results 67
Wing length at 19° and 29°C 67
Sperm viability under osmotic stress 68
Male effects on female fertility 70
Female effects on stored sperm 73
Fertilisation rate 75
Sperm storage 77
Does the decrease in sperm predict female fecundity? 80
Discussion 82
Phenotypic plasticity vs. local adaptation 82
Sperm effects, seminal fluid effects and female effects on fertility 83
Supernumerary spermathecae 87
Conclusion 87
References 89
Chapter 4
Effect of spermathecal proteins on female fertility and sperm storage in Drosophila melanogaster 94
Abstract 94
Introduction 95
Material and Methods 99
Fly Stocks 99
UAS/GAL4 crosses and controls 101
General experimental procedure 103
Experimental fly strains 103
Parameters used to assess sperm storage capability 103
Mating procedure 104
Oviposition and progeny development 104
Part I. RNAi screen (Experiments 1 and 2) 109
Material and methods 109
Experiment 1: Preliminary RNAi screen 109
Experiment 2: RNAi screen 109
Results 110
Experiment 1: Preliminary RNAi screen 110
Experiment 2: RNAi screen 114
Part II: Spermathecal secretory function (Experiments 3 to 5) 121
Material and Methods 121
Experiment 3: Survey of fertility effects of the spermathecal secretory function 121
Experiment 4: Verifying experiment 1 122
Experiment 5: Verifying experiment 2 122
Results 122
Experiment 3: Survey of fertility effects of the spermathecal secretory function 122
Experiment 4: Verifying experiment 1 127
Experiment 5: Verifying experiment 2 128
Part III. Trpa1 and temperature effects 130
Material and Methods 130
Results 131
Sperm number and fertilisation rate 140
Discussion 151
Effect of knock-down of genes with putative role in sperm storage in SSC on fertility 151
Effect of impaired spermathecal secretory function on fertility 153
Effects of enhanced secretory function of the SSC on fertility and sperm storage 154
Wildtype variation in fertility (and sperm storage) with temperature 155
Conclusion 157
References 157
Chapter 5
General discussion 164
Measuring sperm quality in male and female storage organs 164
Environmental effects on sperm stored in males and females 166
Effect of the direct environment on sperm stored in females 168
Conclusion 169
References 170
Acknowledgements 175
Supplementary Material 176
Composition of corn/yeast food 176
Composition of yeast food 176
Chapter 2 176
Chapter 3 177
Full models 177
Survival and mating rate 179
Interaction plots males 181
Fertility of focal males 184
Sex ratio of the progeny of focal males 185
Female fertility 185
Chill coma recovery assay 187
Results 187
Chapter 4 189
Experiment 3 189
Experiment 6 189
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:83712 |
| Date | 23 February 2023 |
| Creators | Eckel, Barbara Angela |
| Contributors | Reinhardt, Klaus, Fricke, Claudia, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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