Chromosomal rearrangements in evolution and in modern mammals: Different analyses and novel techniques for identifying potential fertility problems

Reproductive isolation between species and reproductive problems in individual animals have certain parallels. When a chromosomal rearrangement, (e.g., translocation) occurs, it can cause sub-fertility by impeding meiosis, recombination reduction and chromosomally unbalanced gamete production. In extremely rare occasions, during evolution, such an occurrence can begin the process of reproductive isolation (speciation) if the translocation becomes fixed in the population in homozygous form. Analogies include the common (1:1000) 13:14 Robertsonian chromosome fusion (a cause of infertility) and human chromosome 2, a fusion of two ancestral great ape chromosomes. Chromosomal rearrangements occur more in sperm than eggs and are mediated through Deoxyribonucleic acid (DNA) breakage, another correlate of infertility. There is sufficient concern about sperm DNA damage due to reactive oxygen species (ROS) and lipid peroxidation (LPO) (and their connections to male fertility), that some laboratories are establishing routine DNA damage screening services alongside translocation screening. For any mammal, if fertility is indicated, a routine chromosome analysis and a screen for sperm DNA damage or ROS/LPO would be prudent. Ensuring these services follow accreditation body and best practice guidelines is imperative. Animal breeders and physicians make decisions based on these results and are aware that they could have major impacts on the andrology sector and/or the genetic improvement of a species. To this end, this thesis considers aspects of chromosome rearrangement during evolution and as a cause of infertility, causes of sperm DNA damage and the monitoring and regulation testing in the andrology sector. Specifically, this thesis:

• Assessed a panel of 48 sequence- and conservation score-based homologous cattle fluorescence in-situ hybridisation (FISH) probes in both phylogenetically similar and more distantly related mammals for cross-species hybridization patterns. This was achieved successfully, with a general pattern that the greater the evolutionary distance from cattle, the less successful the probes. For instance, μ = 76.3% of the probes successfully hybridised to the closely related Artiodactyls such as bison, barasingha and sheep, but only μ = 49.3% for the more distantly related non-Artiodactyl species. The potential for these cattle probes to be used to assess chromosome abnormalities in individual species is discussed.

• Assessed equine fertility using cytogenetic analysis and a novel technique for accurate detection of chromosome rearrangements. This was partly achieved based on previous strategies developed in pigs and cattle using sub-telomeric FISH probes. A total of 64.1% Texas Red and 56.3% Fluorescein isothiocyanate (FITC) probes (60.2% total) successfully hybridised to the required locus, leading to the development of a device that was capable of confirming 10 of 19 horse karyotypes, while identifying three novel rearrangements that were previously untraceable by karyotyping alone. The strategy has been employed in the horse breeding community, as has an adapted approach for assessing DNA damage in equine sperm.

• Developed a flow cytometric assay for membrane lipid oxidation in human and equine sperm thereby testing the hypothesis that there are fundamental differences between the species and DNA damage as well as oxidation reduction potential (ORP) and LPO screening could be valid tools for identifying fertility potential of a male. This was successfully achieved, with differences including horses (μ = 11.7%) having a much lower Sperm Chromatin Structure Assay (SCSA®) DNA fragmentation index (DFI) than humans (μ = 21.6%), while the correlation between LPO and DFI for the frozen-thawed human samples (R = -0.380) was much lower than for the frozen-thawed stallion sperm (R = -0.970). A regulatory framework suitable for introduction in the clinic (humans) or specialist testing laboratories (horse) was suggested for DNA damage and ROS screening, yet further work is needed on the LPO test to provide accurate and repeatable results.

• Tested the hypothesis that there are differences in semen analysis protocols between laboratories and clinics based on the regulatory body with which they are accredited and the type of entity they claim to be. Here it was established that United Kingdom Accreditation Service (UKAS) accredited laboratories are more likely to adhere to International Organization for Standardization (ISO) 15189 standards and World Health Organization (WHO) guidelines, than Human Fertilisation and Embryology Authority (HFEA) licensed clinical laboratories. Many laboratories are not demonstrating any internal quality controls or adhering to external quality assurance programs, and there is a clear lack of standardisation within the United Kingdom (UK) andrology sector.

Overall, this thesis was successful in the achievement of its aims with several parallels across the superficially quite diverse, individual chapters drawn. In particular, results appear to substantiate the correlation between stallion sperm's "live fast, die young" analogy of energy production which has recently been associated with their fertility in the literature. This work has made significant contributions towards understanding gross genomic rearrangement (chromosomal and DNA damage) in both reproduction and reproductive isolation. It has developed novel strategies for fertility diagnosis and provided a framework and insight into how this might be regulated.