published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

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No description available.

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Objective markers to identify higher fertility individuals are needed to maximize livestock breeding success. Two heparin-binding proteins, which are reflective of fertility in bulls, have been biochemically identified as fertility-associated antigen (FAA) and tissue inhibitor of metalloproteinases-2 (TIMP-2). These four studies were designed to examine the importance of those proteins in relation to reproduction in bulls and other livestock species. In the first study, indirect immuno-fluorescent microscopy was performed to localize FAA and TIMP-2 to livestock sperm. FAA was localized on spermatozoal acrosomes of bulls and rams, but no cross-reactivity was observed for stallions. TIMP-2 labeling was observed on acrosomes and posterior heads, which was species dependent. Localization patterns for FAA and TIMP-2 were further investigated during heparin-induced capacitation and acrosome reactions of bovine sperm. In study two, an enzyme-linked immunosorbent assay (ELISA) was developed to determine concentrations of FAA in bovine seminal plasma (SP). A commercially available TIMP-2 ELISA was utilized to quantify TIMP-2. Respective mean concentrations of FAA and TIMP-2 in SP were 6.661.487 ug/ml and 1.180.045 mg/ml. Concentrations of FAA in SP did not correspond to bull fertility potential, however, older bulls with higher concentrations of TIMP-2 in SP sired more calves. The third study evaluated utility of an amplified fragment length polymorphism with bovine TIMP-2 gene specific primers to amplify a 700 bp genomic DNA (gDNA) product from sperm. From 53 bulls screened, 22.6% were negative for the 700 bp amplicon. There was a three-fold likelihood for 700 bp negative bulls to not sire a calf compared to 700 bp positive bulls. The product was cloned and sequenced, but no homology to TIMP-2 was detected. Therefore, the product represented novel bovine gDNA sequence. The fourth study identified an equine homologue to the bovine FAA gene. Immuno-based diagnostics had not detected FAA in stallion semen. The equine DNA homologue was 88.5% identical in nucleotide and 86% in amino acid sequences to bovine FAA. Subtle differences in the amino acid sequence are likely responsible for the inability to detect FAA in stallion semen with FAA antibodies to bovine FAA.

Infertility affects ~20% of couples in Europe and in 50% of cases the problem lies with the male. The development of assisted reproductive technologies (ART) such as in vitro fertilisation (IVF) and intra-cytoplasmic spermatozoa injection (ICSI) has allowed some couples to overcome male-factor infertility. However concerns remain over the increasing use of ART as elevated levels of DNA damage in sperm from infertile men have been reported and a link between DNA damage in sperm and early embryonic failure has been demonstrated. DNA damage in sperm, caused by oxidative stress may also be passed on from father to child resulting in an increased incidence of childhood cancer. This has led to fears that the use of damaged sperm in ART could contribute to early embryonic failure and/or birth defects. The studies described in this thesis used mouse models to investigate the relationship between DNA integrity in male germ cells and male fertility. This was achieved by studying both the effects of targeted ablation of genes involved in DNA repair and the impact of scrotal heat stress on testicular function and sperm DNA integrity. Three lines of transgenic mice with deletions in genes involved in genomic integrity (Ercc1, Msh2 and p53) were studied. All three genes are expressed in the testis. These studies confirmed and extended studies on Ercc1 knockout (-/-) mice showing reduced germ cell complement, increased apopotosis, an increased percentage of damaged sperm and demonstrated for the first time that depletion of Ercc1 results in an increased incidence of unrepaired double strand DNA breaks (DSB) in pachytene spermatocytes. The persistence of DSBs in spermatocytes and abnormal sperm chromatin structure confirmed that the repair functions of Ercc1 are essential for normal germ cell maturation. In the p53-/- mice these studies showed for the first time that there was an increase in DSBs in spermatocytes and an increase in numbers of sperm with damaged DNA. The level of apoptosis was also increased in the testes suggesting that caspase-3 mediated apoptosis is not entirely p53 dependent as been previously suggested. These studies demonstrated for the first time that targeted ablation of Msh2 compromises germ cell complement and as in the Ercc1-/- this resulted in gaps in the seminiferous epithelium consistent with clonal loss of germ cells. Consistent with a role for MSH2 in mismatch repair no DSBs were detected in spermatocytes from Msh2-/-. Testicular function is temperature dependant and due to their location in the scrotum testes are normally kept between 2ºC and 8ºC below core body temperature. In mice transient scrotal heat stress (30 minutes at 38°C, 40°C and 42°C) disrupted testicular function. Analysis of sperm and testis parameters revealed that stress at 38°C was sufficient to have subtle effects on epididymal function but the higher temperatures had additional consequences for testicular function which resulted in DNA damage in spermatocytes, germ cells loss and increased apoptosis. Further studies into the pathways of apoptosis demonstrated that the mitochondrial/intrinsic pathway plays a role in heat stress response. The fertility of males was altered in those heated to 42°C resulting in reduced pregnancy rate and litter size. Given that the paternal genome is reported to be required for the development of extraembryonic tissues and this will influence growth of the embryo, it was interesting to note an increase in resorption sites in pregnancies using 40°C males. IVF was used to demonstrate that embryos formed using sperm from males stressed at 42°C were compromised between the 4-cell and blastocyst stage suggesting that though sperm with DNA damage are still capable of fertilisation, the paternal DNA was introducing genomic instability to the embryo and having fatal effects on development. These studies have also shown that one possible underlying cause of the disturbance in testicular function is hypoxia, as a marked increase in Hif1 alpha (a marker of hypoxia) mRNA and relocalisation of the protein was observed in the testis. In conclusion, DNA damage in the male germ line caused either by induced stress, or by targeted ablation of DNA repair genes, can disrupt testicular architecture, function and therefore the fertility of mice. These data have demonstrated that deletion of Ercc1, Msh2 and p53 can have differential but overlapping affects on germ cell function and sperm production and that increased scrotal temperature can cause subfertility in male mice. This study has provided further confirmation of possible male-mediated effects on embryo survival and these findings should be taken into consideration when using sperm from infertile men in IVF/ICSI treatments where the normal quality control processes involved in fertilisation are bypassed.

A nivel mundial se está dando un fenómeno que cada vez es más común, la infertilidad. En la actualidad la edad es considerada un factor determinante en la calidad seminal, existe una relación directa entre la edad y el aumento del daño en el ADN espermático. El objetivo de este estudio fue evaluar las características seminales en jóvenes universitarios mediante espermatogramas utilizando el sistema computarizado de análisis seminal C.A.S.A (Computer Assisted Sperm Analyzer, ISAS v1.2) para la evaluación morfológica. Se calcularon los estadísticos descriptivos, frecuencias y coeficientes de variación para todos los parámetros seminales procedentes de 30 jóvenes universitarios voluntarios de 18 a 30 años de edad. Con el fin de determinar si los hábitos de los jóvenes consideradas en la investigación tuvieron efecto en alguno de los parámetros seminales se realizó la prueba exacta de Fisher, en el caso de variables nominales, y una prueba de T de student o de U de Mann Whitney, previa verificación de la normalidad con la prueba de Shapiro-Wilk, en el caso de las variables cuantitativas. Todos los análisis se realizaron con un nivel de confianza de 95% en el software SPSS v.21. En conclusión no se encontraron asociaciones significativamente estadísticas entre los hábitos y los distintos parámetros seminales y se determinó que solo los criterios de pH, volumen, vitalidad, motilidad, concentración y recuento total, cumplen con los valores establecido por la Organización Mundial de Salud y la Sociedad Europea de Reproducción Humana y Embriología (ESHRE), a diferencia del 76.7% las muestras seminales que no cumple con los criterios de morfología. Se observó que existían alteraciones morfológicas en la cabeza y la pieza intermedia de los espermatozoides, comparándolo con los valores considerados normales por la OMS se obtuvo que las principales áreas afectadas fueron la longitud, el ancho, el área, la elipticidad y la elongación de la cabeza al igual que el ancho de la pieza intermedia, y al compararlo con los valores de la ESHRE se obtuvo que las principales áreas afectadas fueron el ancho, el área y la elipticidad de la cabeza. Worldwide is taking a phenomenon that is becoming more common, infertility. Today's age is considered a important factor in semen quality, there is a direct relationship between age and the increased damagein DNA sperm. The aim of this study wasto evaluate the seminal characteristics in young students by Spermograms and use a computerized semen analysis C.A.S.A (Computer Assisted Sperm Analyzer, ISAS v1.2) for morphological evaluation Descriptive statistics and frequencies for all semen parameters considered in this study were calculated. The coefficient of variation was calculated by dividing the standard deviation to the average and expressed in percentage. In order to determine whether the habits of young people considered in the investigation had any effec on semen parameters Fisher's exact test was performed, in the case of nominal variables, and student T test or Mann Whitney, after verification of normality with the Shapiro-Wilk test, in the case of quantitative variables. All analyzes were performed with 95% confidence in the SPSS v.21 software In conclusion not significantly statistical associations between the habits and different sperm parameters were found and determined that only the criteria of pH, volume, vitality, motility, concentration and total count, meet values set by the World Health Organization and the European Society of Human Reproduction and Embryology, unlike 76.7% of young people who do not meet the requirements criteria of morphology. It was found that there were obvious morphological changes in the anatomical parts of the head and the intermediate piece, comparing the valuesconsidered normal by WHO, was obtained that the main affected areas were the length, width, area, ellipticity and elongation of the head as the width of the intermediate piece, and when compared with the valuesthat were obtained ESHRE relevant affected areas were the wide area and the ellipticity of the head.