The role of immune-mediated female sperm selection in temporal dynamics of fertilisation bias

Lead Research Organisation: University of Oxford
Department Name: Zoology

Abstract

The way paternity is distributed across males has considerable impact on the ecology and evolution of a population, regulating the effective population size and the amount of standing genetic variance. Despite intense selection on reproductive success paternity remains highy variable, and understanding the mechanisms underpinning this variation is a fundamental challenge in Biology. Increasing evidence indicates that an important source of variation in paternity originates from processes occurring after insemination. In most organisms the ejaculates of multiple males often compete to fertilise a set of eggs, and females can drastically influence the outcome of this competition through biased responses to the sperm of different males. Females are expected to bias fertilisation in favour of males of higher genetic quality in order to increase the success of their offspring. Because the genetic diversity (heterozyosity) of an individual promotes survival, a increasingly topical hypothesis is that females preferentially utilise the sperm of males that are genetically different from the female to promote offspring heterozygosity. However, evidence for this hypothesis is ambiguous and -contrary to theory- females may often bias fertilisation in favour of genetically similar rather than dissimilar males. Recent evidence from different species including our own work in the red junglefowl indicates that this counterintuitive response may be regulated by genetic similarity at the Major Histocompatibility Complex (MHC). The MHC is a complex of genes that play a fundamental role in immune responses allowing the organism to recognise self from non-self and respond against cells that are not recognised as self. While this immune response enables the organism to combat pathogens and parasites it may also result in a side-effect differential response to sperm of different males. Namely, we expect the female immune system to tolerate sperm of males that share MHC genes with the female and discriminate against the sperm of males that have a different MHC profile. As it is typical of similar immune responses, we also expect female response to the sperm of a certain MHC similarity with the female to change as the female is exposed to successive inseminations with the same type of sperm. It is plausible that, through continued exposure, the female immune system 'learns' to recognise MHC-similar sperm type, thus reducing the bias in paternity. This novel hypothesis is founded on well established immunological mechanisms and represents a biologically plausible proximate explanation consistent with an emergent trend of studies indicating that fertilisation may be biased in favour of genetically similar partners. Elucidating the consequences of MHC-mediated immune responses for female sperm selection would therefore contribute to unravel the mechanisms underpinning variation in paternity in natural populations. The aim of the proposed research is to test experimentally different key predictions of the hypothesis that fertilisation is biased by MHC-mediated immunological responses of the female reproductive tract to the ejaculates of different males. We will test these predictions in a well characterised population of red junglefowl. Red junglefowl are the wild ancestor of the domestic chicken and an ideal system to study MHC-mediated female sperm selection. First, females typically obtain ejaculates from multiple males and are known to bias fertilisation in different ways, including in favour of MHC-similar sperm. Second, poultry techniques of artificial insemination and sperm assays enable us to study post-insemination processes non-invasively and under controlled conditions. Third, the MHC of the fowl is very simple and extremely well characterised. Finally, we have a deep understanding of the mechanisms that modulate paternity skews in this species, including the role of MHC similarity.

Publications

10 25 50
publication icon
Alonzo SH (2013) Selection on female remating interval is influenced by male sperm competition strategies and ejaculate characteristics. in Philosophical transactions of the Royal Society of London. Series B, Biological sciences

publication icon
Carleial R (2020) Temporal dynamics of competitive fertilization in social groups of red junglefowl (Gallus gallus) shed new light on avian sperm competition. in Philosophical transactions of the Royal Society of London. Series B, Biological sciences

publication icon
Collet J (2012) Sexual selection and the differential effect of polyandry. in Proceedings of the National Academy of Sciences of the United States of America

publication icon
Collet JM (2014) The measure and significance of Bateman's principles. in Proceedings. Biological sciences

publication icon
Cornwallis CK (2014) Sex-specific patterns of aging in sexual ornaments and gametes. in The American naturalist

publication icon
Dean R (2011) The risk and intensity of sperm ejection in female birds. in The American naturalist

 
Description The overall goal of the current project licence is to determine the role of female immunity in sperm selection and fertilisation bias in a population of red junglefowl, Gallus gallus. Specifically, the project seeks to address the following objectives:
1. To investigate patterns of Major Histocompatibility Complex (MHC)-dependent fertilisation bias and temporal dynamics arising over experimental repeated female exposure (indicative of acquired responses).
2. To establish which immune responses are induced in the female reproductive tract by insemination.
3. To test the effect of immune manipulation on MHC-dependent anti-sperm responses.
4. To determine candidate sperm-expressed MHC-linked target(s) under immune selection.
The project is producing substantial experimental results against each one of these objectives, which collectively represent a considerable contribution to our understanding of how female immune responses impact fertilisation dynamics in vertebrates.
1. Our results indicate that similarity at the MHC between a male and a female is an important predictor of fertilisation success only under certain conditions, namely: (a) to the extent to which variation in MHC similarity predicts genome-wide genetic similarity (i.e. relatedness) between individuals; and (b) particularly when females are exposed to their partners (as opposed to being artificially inseminated). These results suggest that MHC similarity can play a role in inbreeding avoidance when females are exposed to phenotypic cues of the male. These results are partly published (Løvlie et al. 2013 Proc. Roy. Soc. Lond. B) and partly in process of being submitted for publication. which has paved the way and informed further experimental work on the study population. A combination of in vitro assays and invivo experiments (through artificial insemination) is providing evidence that female sperm utilisation is inherently and repeatedly biased towards specific males, and that cumulative sperm exposure strongly changes such bias over time. These temporal dynamics are complex: females that don't respond strongly against the sperm of a particular male tend to mount a progressively stronger response against his sperm over successive weeks of exposure to the sperm of this male, while females that display a strong initial response against the sperm of a male tend to become more tolerant of his sperm over time. We have further shown that these temporal dynamics predict the outcome of fertilisation when the sperm of a male immunologically familiar to a female compete with the sperm of an immunologically novel male. We are in the process of consolidating these results, refining analysis of the data collected so far and planning follow-up empirical work. So far, there is no evidence that these effects are caused by MHC similarity and indicate that both acquired and innate immune mechanisms are involved (see below).
2. We have developed a range of in vitro assays to demonstrate that the in vivo patterns described above are determined by two distinct responses by acquired and innate immunity. Acquired. We have detected that in vitro sperm exposure to female blood plasma leads to rapid agglutination of sperm cells and a drastic reduction in the swimming velocity of non-agglutinated sperm. These effects persist even at very low titrations, and are repeatable and differential for individual male:female combinations indicating that some females respond consistently more strongly to the sperm of certain males. These biases arise in virgin females following the onset of egg laying, indicating that sperm bias are inherent and triggered by sexual maturity. Using a combination of ELISAs, Western Blots, and heat denaturation assays we have shown that these responses are mediated by antibodies, specifically IgM. Importantly, the in vitro response of female plasma to the sperm of a male predicts the patterns of sperm utilisation by a female following artificial insemination by the same sperm donor, confirming that the in vitro assay can be used as predictive tool of in vivo responses. Work based on immunoprecipitation and proteomics is seeking to identify the sperm antigens mediating this acquired response, and their potential polymorphism in the study population. Innate. We have shown that chicken macrophages engulf live sperm cells through phagocytosis in vitro, and that the sperm of certain males trigger a macrophage response that is consistently stronger (more sperm engulfed faster) across females that the sperm of other males. In other words, variation in innate responses appear determined by male effects rather than by male:female interactions (unlike the acquired responses outlined above). Further work is on-going to identify the specific mechanisms mediating this response. Preliminary results indicate that Pattern Recognition Receptors are involved in the recognition of sperm antigens. More specificially, it would appear that recognition is mediated by avian Toll-Like Receptor 15 (TLR15), possibly triggered by hyaluronic acid generated by the enzymatic activity of sperm cells. We are in the process of consolidating these results, refining analysis of the data collected so far and planning follow-up empirical work.
3. We have conducted a vaccination experiment in which females were immunised with the sperm of a particular donor, to show that vaccination had a profound impact on temporal patterns of sperm utilisation, providing further confirmation of the involvement of acquired responses. Preliminary results show that in vivo patterns of female sperm utilisation are influenced by the genetic relatedness between the female and the sperm donor. We are in the process of consolidating these results, refining analysis of the data collected so far and planning follow-up empirical work.
4. An experiment in which semen from MHC-heterozygous males was inseminated in MHC- homozygous females revealed no evidence of female selection of sperm based on sperm MHC haplotype. We are conducting sperm proteomic work to identify the sperm antigens triggering both acquired and innate responses (see above).
Progress in this project in the last 12 months has been severely hampered by the pandemic.
Exploitation Route The results of this project open up a range of novel avenues of research. Two particularly important ones are:
1. Identify the sperm antigens mediating female acquired responses to sperm, and determine the mechanisms that predispose virgin females to respond against certain males more than others, including genetic polymorphism in the population and cross-reactivity across sperm and non-sperm (e.g. pathogen) antigens.
2. Establish the role of Pattern Recognition Receptors and TLR15 in the innate recognition of sperm antigens.
Sectors Agriculture, Food and Drink

 
Description The findings of this project have contributed to 35 scientific publications, and several additional publications are at different stages of development. Overall this research has the potential to inform selection and management of commercial poultry with positive impact on animal health and welfare, biosecurity and the efficiency of the poultry meat food system. So far, these findings have contributed to inform several outreach and engagement activities, and have been disseminated in seminars and lectures.
First Year Of Impact 2013
Impact Types Cultural,Economic

 
Description ASAB research grant
Amount £4,934 (GBP)
Organisation University of Nottingham 
Sector Academic/University
Country United Kingdom
Start  
 
Description Clarendon Scholarship
Amount £42,000 (GBP)
Organisation University of Oxford 
Sector Academic/University
Country United Kingdom
Start 09/2013 
End 09/2016
 
Description Industrial LINK Grant
Amount £415,155 (GBP)
Funding ID BB/L009587/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2014 
End 04/2017
 
Description Marie Curie Fellowship
Amount £281,257 (GBP)
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 05/2012 
End 09/2014
 
Description NSERC Postdoctoral Fellowship
Amount $80,000 (CAD)
Organisation Natural Sciences and Engineering Research Council of Canada (NSERC) 
Sector Public
Country Canada
Start 01/2011 
End 09/2013
 
Description Natural Motion Scholarship
Amount £44,000 (GBP)
Organisation University of Oxford 
Sector Academic/University
Country United Kingdom
Start 09/2017 
End 09/2019
 
Description PhD Scholarship to study fertility in fowl populations
Amount £60,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2019 
End 09/2023
 
Description Royal Society International Incoming Short Visits
Amount £4,500 (GBP)
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start  
 
Title red junglefowl semen samples 
Description Collection of several hundreds of samples comprising centrifuged seminal fluid obtained from natural matings of a study population of red junglefowl 
Type Of Material Biological samples 
Year Produced 2018 
Provided To Others? No  
Impact Initiation of collaboration with Syracuse University to characterise seminal fluid proteomics 
 
Title Data from: Temporal dynamics of competitive fertilization in social groups of red junglefowl (Gallus gallus) shed new light on avian sperm competition 
Description Studies of birds have made a fundamental contribution to elucidating sperm competition processes, experimentally demonstrating the role of individual mechanisms in competitive fertilisation. However, the relative importance of these mechanisms and the way in which they interact under natural conditions remain largely unexplored. Here, we conduct a detailed behavioural study of freely-mating replicate groups of red junglefowl, Gallus gallus, to predict the probability that competing males fertilise individual eggs over the course of 10-day trials. Remating frequently with a female and mating last increased a male's probability of fertilisation, but only for eggs ovulated in the last days of a trial. Conversely, older males, and those mating with more polyandrous females, had consistently lower fertilisation success. Similarly, resistance to a male's mating attempts, particularly by younger females, reduced fertilisation probability. After considering these factors, male social status, partner relatedness and the estimated state of a male extragonadal sperm reserves did not predict sperm competition outcomes. These results shed new light on sperm competition dynamics in taxa such as birds, with prolonged female sperm storage and staggered fertilisations. 
Type Of Material Database/Collection of data 
Year Produced 2020 
Provided To Others? Yes  
URL http://datadryad.org/stash/dataset/doi:10.5061/dryad.cz8w9gj1d
 
Title Red junglefowl reproductive microbiome 
Description Large dataset comprising the operational taxonomic units identified through 16S sequencing of samples from different regions of the reproductive tract and ejaculates of the study population. 
Type Of Material Database/Collection of data 
Year Produced 2020 
Provided To Others? No  
Impact Collaboration with Prof Tom Bell (Imperial College) and Dr Melissa Rowe (Netherlands Institute of Ecology, NIOO) to study reproductive microbiota in bird species 
 
Title sperm agglutination 
Description These datasets contain: (a) the results of a series of experiments characterising variation in in vitro sperm agglutination to blood serum in junglefowl and domestic chickens and patterns of female sperm utilisation in vivo following insemination, including the results of experimental immunisation work; and (b) the results of immunological tests investigating the proximate mechanisms of differential sperm agglutination, e.g. through ELISA-based in vitro work. 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact This information is in the process of being analysed and submitted for publication. Aspects of these data and results have been presented at workshops and conferences. 
 
Title sperm allocation dataset 
Description This dataset contains information on mating behaviour and ejaculate characteristics for natural ejaculates produced by male junglefowl under different experimental conditions. The dataset is partly published in open access (Alvarez-Fernandez et al. 2019 Sci Reports; Borziak et al. 2016 Sci Reports). More recent parts of the dataset are in the process of being analysed for future publication. 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
Impact Expression of interest from academics and members of commercial poultry companies. Request of new collaborations. 
URL https://www.nature.com/articles/s41598-019-41336-5
 
Title sperm:macrophage interaction 
Description This dataset contains the results of in vitro experiments investigating the way chicken macrophage respond to sperm and sperm agglutinates, and proximate mechanisms underpinning sperm-driven macrophage activation. 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact These results are being developed for scientific dissemination. Some results have already been presented in talks, conferences and workshops. 
 
Description Collaboration with Prof Bernd Kaspers, Institut für Tierphysiologie, University of Munich, Germany 
Organisation Ludwig Maximilian University of Munich (LMU Munich)
Department Faculty of Psychology and Educational Sciences
Country Germany 
Sector Academic/University 
PI Contribution This collaboration seeks to harness the power of chicken genetic knockout lines developed in Munich to establish the causal effect of antibody response in sperm agglutination.
Collaborator Contribution Access to knock-out birds (homozygotes and heterozygote controls) for experimental work.
Impact This collaboration is expected to make a key contribution to future publications.
Start Year 2017
 
Description Collaboration with Prof Tom Bell (Imperial College) and Dr Melissa Rowe (Netherlands Institute of Ecology, NIOO) to study reproductive microbiota in bird species 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Development of sampling protocols, experimental work and conceptual leadership of the collaboration
Collaborator Contribution Supervision of the 16S sequencing pipeline (Imperial), conceptual and technical input (NIOO)
Impact Feature review published in Trends Ecology & Evolution
Start Year 2018
 
Description Collaboration with Prof Tom Bell (Imperial College) and Dr Melissa Rowe (Netherlands Institute of Ecology, NIOO) to study reproductive microbiota in bird species 
Organisation Netherlands Institute of Ecology
Country Netherlands 
Sector Charity/Non Profit 
PI Contribution Development of sampling protocols, experimental work and conceptual leadership of the collaboration
Collaborator Contribution Supervision of the 16S sequencing pipeline (Imperial), conceptual and technical input (NIOO)
Impact Feature review published in Trends Ecology & Evolution
Start Year 2018
 
Description Collaboration with Prof. Jim Kaufman (Cambridge University and Edinburgh University) for Initiation of a collaboration with Prof. Jim Kaufman (Cambridge University and Edinburgh University) for PCR-NGS typing the Major Histocompatibility Complex of the red junglefowl 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution Contribution to coordination of the collaboration from conceptual to logistic. Provision of biological samples and biological information.
Collaborator Contribution Application a newly developed PCR-NGS typing approach.
Impact The collaboration has only just started.
Start Year 2020
 
Description GoCAS workshop on Sexual Selection and Sexual Conflict, University of Gothenburg, Sweden. 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Workshop bringing together an international panel of experts to clarify outstanding issues in the field of sexual selection. The workshop resulted in a join publication in PeerJ.
Year(s) Of Engagement Activity 2017
URL https://peerj.com/articles/7988/
 
Description Organiser of Workshop "Animal Health, Fertility and Welfare in Poultry Productions" 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact The workshop brought together academics, industry and people engaged with policy making to disseminate findings of our NERC-funded research and discuss broader implications for animal production, health and welfare in poultry from different perspectives (industry, academia, policy making).
Year(s) Of Engagement Activity 2014
 
Description Plenary speaker at LVI Symposium Poultry Scientific Symposium of Spanish Poultry Science Association - World's Poultry Science Association (AECA-WPSA) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Plenary presentation detailing the role of basic and applied research in our lab in elucidating mechanisms underpinning fertility for commercially important populations. The meeting was attended by poultry scientists, veterinarians and commercial companies from different countries.
Year(s) Of Engagement Activity 2019
URL https://56symposiumavicultura.com/es/Inicio
 
Description Plenary talk at Graduate Symposium of the Zoology Department of Stockholm University 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact A plenary presentation of basic and applied work in our research group aimed at unravelling mechanisms underpinning fertility in poultry. The audience comprised graduate students, academic researchers, a number of donors from different countries.
Year(s) Of Engagement Activity 2019
 
Description Summer School outreach activities 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact UNIQ Summer school activity by members of the group to introduce pupils from secondary schools to our research.
Year(s) Of Engagement Activity 2017,2018,2019
URL http://www.uniq.ox.ac.uk/c/biology
 
Description Talk at local primary school (S.S. Mary and John, Oxford) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact ~50 pupils and several teaching staff attended this
Year(s) Of Engagement Activity 2016
 
Description UNIQ Summer School 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Schools
Results and Impact The work feature in a UNIQ Summer School event dedicated to Evolution through presentation and activities organised by Dr Cedric Tan, PDRA on the grant.

Increased awareness of the reproductive behaviour and physiology of a commercially significant organism.
Year(s) Of Engagement Activity 2013
 
Description participation in the BBC documentary Attenborough's Wonder of Eggs (BBC 2) 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Our study population of red jungefowl was featured and our research on the reproductive biology of this species informed the narrative of this part of the documentary
Year(s) Of Engagement Activity 2018
URL https://www.bbc.co.uk/programmes/p062qkqy