General consensus agrees that the oscillations in intracellular calcium (Ca2+) leading to oocyte activation are induced by a testis-specific phospholipase C, PLCzeta (PLCS). Strong evidence now indicates that certain types of human infertility are caused by failure of the sperm to activate the oocyte in an appropriate manner. Such sperm exhibit absent/reduced levels of PLCS, while molecular analysis of the PLCS gene in an infertile patient identified a substitution of a conserved histidine residue at position 398 of the PLCS protein sequence for a proline residue (H398P), leading to severely reduced PLCS function and oocyte activation efficiency. Collectively, these studies indicate the therapeutic and diagnostic potential use of PLCs for assisted reproductive technology (ART). In the present study, a novel mutation was identified in the patient exhibiting the H398P mutation, which caused histidine to be substituted for leucine at position 233 (H233L) of the PLCS protein sequence. The H233L mutation severely reduced PLCS functional ability, and investigation of the inheritance patterns of H233L and H398P revealed that the patient inherited H233L from his mother and H398P from his father. Additionally, data suggested that quantitative immunofluorescence of PLCS in human sperm may not be an informative indicator of oocyte activation capability, while proportional analysis of sperm exhibiting PLCS immunoreactivity may reflect fertility. Density gradient washing (DGW) increased the proportion of human sperm exhibiting PLCS immunofluorescence, while cryopreservation reduced total PLCS immunofluorescence within fertile human sperm. While recombinant human PLCS protein was successfully purified from a prokaryotic system, this proved to be inactive following mouse oocyte micro injection. Most importantly, the current study describes for the first time, the successful production of active recombinant human PLCS protein within lysates from transfected transformed human embryonic kidney (HEK293T) cells, which induced characteristic patterns of Ca2+ release when injected into mouse oocytes. The present data indicate that wild type PLCS was transcribed in an active state within a eukaryotic system, while loss-of-activity PLCS isoforms may contribute to overall protein instability, possibly resulting in increased rates of protein degradation. It is demonstrated that PLCS isoforms were localised to the endoplasmic reticulum (ER), with the EF- hand domain possibly determining the localisation of the enzyme. Collectively, these results not only assist with the therapeutic application of PLCs, but also illustrate the complex nature of the mechanisms underlying its functional activity.

Chromosome copy number aberrations are a leading cause of birth defects, stillbirths, pregnancy loss and infertility. Every human male has a proportion of chromosomally abnormal sperm however conditions such as infertility, cancer, cancer treatments, and environmental factors can increase this. Chromosome abnormality is commonplace in human embryos and one reason for the development of the controversial preimplantation genetic screening CPOS). Factors such as embryo quality and maternal age are cornmon correlates. Appropriate nucleus positioning of chromosome territories is also though to be indicative of a "healthy" nucleus with aberrations in such nuclear organization associated with disease. The purpose of this study was to provide insight into the relationship between chromosome copy number, nuclear organization and various aetiological factors in human sperm and early stage embryos. Specifically. • To investigate the nuclear positioning oftelomeric and sub telomeric region in sperm cells and test the hypothesis that such organisation is altered in infertile males. • To investigate the nuclear positioning of centromeric and locus specific regions of 5 chromosomes in sperm cells from males undergoing chemotherapeutic treatment for testicular cancer and Hodgkin's lymphoma and test the hypothesis that either the cancer, or its treatment significantly alters patterns of nuclear organization. • To analyse FISH based PGS and "follow up" in 250 treatment cycles to investigate levels of aneuploidy false negative and positive results, also well as effects of different indications such as maternal age. • To investigate the levels of aneuploidy for all 24 chromosomes using a newly developed multicolour FISH technique. To test hypotheses that factors e.g. maternal age and embryo morphology significantly effect levels, and that day 3 and day 5 results are concordant. • To assess levels nuclear organisation of human embryos for loci on all 24 chromosomes and their relationship to maternal age, day 3 and day 5 embryo morphology. Overall, results provide some evidence for differences in nuclear organisation in infertile males compared to controls for telomeric but not sub-telorneric loci. Effects of cancer (testicular cancer and Hodgkin's lymphoma) and chemotherapy were subtle at best with one testicular cancer patient showing a significant difference compared to controls. In embryos, monosomy appeared more common that trisomy and effects of maternal age and embryo quality were apparent when a small subset of chromosomes were analysed. Similar analysis with a 24 FISH assay confirmed monosomy/trisomy ratios however failed to show significant relationship with maternal age and embryo morphology, thereby raising questions about the reliability of the technique. Finally comparison of various parameters and nuclear organization revealed consistent alterations of the position of specific centromeres (e.g. for chromosomes 3 and 4). In conclusion, FISH is now clearly old technology for PGS but has great potential for the analysis of mosaicism and nuclear organisation.

One in six couples wishing to start a family are infertile. The many causes of infertility include genetic defects that can be single gene, multifactorial or chromosomal (including Y deletions, karyotype abnormalities and gamete aneuploidy). This thesis is concerned with the association between infertility and increased sperm aneuploidy. Specific questions are: should males be screened for sperm aneuploidy before intracytoplasmic sperm injection (ICSI)? Is there a relationship between individual semen parameters and sperm aneuploidy for specific chromosome pairs? What is the role of genome organisation in male gametes and its association with infertility? Whether use of alternative therapy (in this case, traditional Chinese Medicine (TCM)) can be used to improve sperm disomy levels. Statistical analysis of questionnaire data revealed that infertility specialists believed there to be merit in screening sperm aneuploidy levels before ICSI. Evidence is presented for possible chromosome-specific and semen parameter specific mechanisms for sperm aneuploidy as is evidence of genome organisation that may be perturbed in infertile males. Finally, in six males studied, sperm aneuploidy levels improved significantly coincident with TCM. Closer investigation of the biological activity of individual therapeutic herbs and treatment cocktails revealed strong anti-oestrogenic and anti-oxidant properties. This suggests a possible mechanism of action of the herbs and provides the basis from which future placebo controlled clinical trials might continue. Possible criticisms of the work presented here include the unavailability of blood samples from many of the patients (thus preventing karyotype analysis) and the absence of a second control group in our studies on semen parameters. Nevertheless significant steps have been made towards establishing the need for, and the implementation of, a pre-ICSI screening test. Moreover progress has been made towards further understanding the aetiology of sperm aneuploidy and towards the implementation of a new treatment that may, ultimately, augment, or even replace ICSI.