Gamete proteomic profile of male patients suffering from sexually transmitted infections

Flint, Margot Gwyneth (2016-03)

Thesis (PhD)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: The aim of this study was to provide better insight into the effects of Neisseria gonorrhoea, Chlamydia trachomatis, and Trichomonas vaginalis on semen characteristics. In addition, a crucial focus was also to determine the protein profile of spermatozoa isolated from these infected semen samples. Identification of semen samples positive for bacterial colonisation of N. gonorrhoea and T. vaginalis were done through the use of differential isolation mediums. For the detection of samples positive for C. trachomatis, the ImmunoComb® was employed, which is a quantitative indirect enzyme immunoassay. Macro- and microscopic semen and spermatozoa parameters were assessed, including: volume, pH, viscosity, concentration, motility, morphology, viability, acrosome reaction, leukocyte count, and PMN-elastase concentration. To assess prostate and seminal vesicle functioning, the seminal concentrations of citric acid and fructose were photometrically quantified. The quantification of the level of reactive oxygen species (ROS) production was determined by means of flow cytometry and the DNA fragmentation was detected using a commercially available assay. For proteomic analysis, samples were iTRAQ (isobaric tags for relative and absolute quantification) labelled and underwent liquid chromatography-mass spectrometry, followed by data analysis, protein identification, and quantification. Results of the study showed that amongst the three microorganisms, the most prevalent occurrence rate in the population tested was that of T. vaginalis. The effects of the studied sexually transmitted infections (STIs) on the spermatozoa parameters demonstrate the negative impact of the microorganisms on the fertility outcome of the male partner. The study has shown the relationship between leukocyte-derived ROS and DNA fragmentation, which can significantly impair the fertility outcome. Additionally, the findings of decreased concentrations of fructose in the ejaculates positive for N. gonorrhoea, demonstrates the effect of an STI on the glandular functioning of the secretory activity of the seminal vesicles. The observed negative correlation between ROS and DNA fragmentation in samples positive for N. gonorrhoea demonstrates the impact of a pro-oxidant overload on spermatozoa DNA integrity. A considerable number of 178 differentially expressed proteins (DEPs) were identified in the STI positive sample groups. The study subsequently focused on specific proteins according to their role in male fertility. Insight into the role DEPs may play in spermatozoa metabolism and the impact on the motility was provided. Amongst structural related proteins, the down-regulation of outer dense fibre 2, in samples positive for C. trachomatis, was evaluated. Oxidative stress related proteins included the up-regulated superoxide dismutase 1 in the ejaculates positive for N. gonorrhoea and down-regulated peroxiredoxin 5 in the C. trachomatis positive group. From the proteins involved in the response to physiological stress, heat shock proteins was discussed, with the down-regulation of the 70-kDa heat shock protein and the up-regulated HSP 90-kDa-beta member 1, both identified in samples positive for N. gonorrhoea. Among immune response proteins, prolactin-inducible protein (C. trachomatis) was found to be down-regulated, while azurocidin (N. gonorrhoea) and filamin-B (T. vaginalis) were up-regulated. Proteins involved in DNA condensation included down-regulated protamine-2 (C. trachomatis). The identification of proteins, which are differentially expressed between spermatozoa from samples positive for STIs, can provide crucial insight into their possible influence on male fertility and role as potential biomarkers for further research. Research on a proteomic and molecular level could allow for the prevention of the long-term obstacles facing partners experiencing compromised fertility. Despite the ongoing research focusing on the sperm proteome, it can be stated with reasonable certainty, that this is the first study into the proteomic profile and corresponding parameters of spermatozoa isolated from semen samples positive for STIs.

AFRIKAANSE OPSOMMING: Die doel van hierdie studie was om ‘n beter insig te kry rakende die effekte van N. gonorree, C. trachomatis en T. vaginalis op semen eienskappe. ʼn Bykomende fokus was ook om die proteïen profiel van hierdie spermatozoa, wat geïsoleer is vanuit die besmette semen monsters, te bepaal. Semen monsters positief vir bakteriële kolonisasie van N. gonorree en T.vaginalis is geïdentifiseer met behulp van verskillende differensiële isolasie mediums. ‘n Kwantitatiewe indirekte ensiem immunoassay, die ImmunoComb®, was gebruik vir die identifikasie van monsters positief vir C. trachomatis. Makro- en mikroskopiese semen en spermatozoa parameters was geassesseer, insluitend: volume, pH, viskositeit, konsentrasie, motiliteit, morfologie, vitaliteit, akrosoom reaksie, leukosiet telling en PMN-elastase konsentrasie. Seminale vesikel en prostaat funksies is deur middel van fotometriese kwantifikasie van die seminale konsentrasies van sitroensuur en fruktose bepaal. Reaktiewe suurstof spesies (ROS) vlakke is met behulp van vloeisitometrie gekwantifiseer en ‘n kommersiële beskikbare toets was gebruik vir die deteksie van DNA fragmentasie. Vir proteomiese analise doeleindes is monsters met iTRAQ (isobariese merkers vir relatiewe en absolute kwantifikasie) gemerk, waarna dit vloeistofkromatografie- massa spektrometrie ondergaan het. Dit is daarna opgevolg deur data analise and proteïen identifikasie asook kwantifikasie. Die resultate van die studie toon aan dat T. vaginalis die mees algemeenste van die drie mikro-organismes was wat voorgekom het in die populasie wat getoets is. Die effek van die bestudeerde seksueel oordraagbare infeksies op spermatosoa parameters dui die negatiewe impak van hierdie mikro-organismes op die fertiliteit van die man aan. Die studie dui ook die verhouding aan tussen leukosiet afkomstige ROS en DNA fragmentasie, wat vrugbaarheid aansienlik kan benadeel. Daarbenewens dui die bevinding van verlaagde fruktose konsentrasies in die ejakulate positief vir N. gonorree ook daarop dat hierdie seksueel oordraagbare siekte die sekretoriese aktiwiteit van die seminale vesikels beïnvloed. Die waargenome negatiewe korrelasie tussen ROS en DNA fragmentering, in monsters positief vir N. gonorree toon ook die impak van 'n pro-oksidant oorlading op die DNA integriteit van spermatosoa. Die aansienlike getal van 178 differensieel uitgedrukte proteïene (DEPs) is in die STI positiewe groepe geïdentifiseer. Hierdie studie het gevolglik Stellenbosch University https://scholar.sun.ac.za v gefokus op spesifieke proteïene volgens die rol wat hul vervul in manlike vrugbaarheid. Insig in die rol wat DEPs speel in spermatosoë metabolisme en die impak daarvan op motiliteit is gebied. Onder die strukturele verwante proteïene is die af-regulering van buitenste digte vesel 2, in monsters positief vir C. trachomatis, bespreek. Oksidatiewe stres verwante proteïene sluit in die op-gereguleerde superoksied dismutase 1 in die ejakulate positief vir N. gonorree en die af-gereguleerde peroxiredoxin 5 in die C. trachomatis positiewe groep. Van die proteïene wat betrokke is by die reaksie op fisiologiese stres is die volgende hitte-skok proteïene (HSP) geïdentifiseer in monsters positief vir N. gonorrhee: die 70-kDa HSP is af-gereguleer terwyl die HSP 90-kDa-beta lid 1 op-reguleer was. Onder die immuunrespons proteïene is die prolaktien-induseerbare proteïen (C. trachomatis) bevind om af-gereguleerd te wees, terwyl azurocidin (N. gonorree) en filamin-B (T. vaginalis) op-gereguleerd was. Proteïene betrokke by DNA kondensasie sluit in die af-gereguleerde protamien-2 (C. trachomatis). Die identifikasie van proteïene wat differensieel uitgedruk word tussen spermatosoa van monsters positief vir seksueel oordraagbare siektes kan noodsaaklike insig verskaf rakende hul moontlike invloed op manlike vrugbaarheid en kan potensieel gebruik word as biomerkers. Navorsing op 'n proteomiese en molekulêre vlak kan voorsiening maak vir die voorkoming van die langtermyn-struikelblokke vir lewensmaats wat vrugbaarheidsprobleme ervaar. Ten spyte van die voortdurende navorsing wat fokus op die sperm proteoom, kan dit met redelike sekerheid gestel word dat dit die heel eerste omvattende studie is met betrekking tot die proteomiese profiel en ooreenstemmende parameters van spermatosoa geïsoleerd vanuit semen monsters positief vir seksueel oordraagbare siektes.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/98639
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