Presence of apoptotic bodies in the human ejaculate: effects of exposure to persistent organochlorine pollutants

Microorganisms play a central role in the regulation of ecosystem processes, and they comprise the vast majority of species on Earth. The aim of this PhD thesis was to improve our knowledge on microbial diversity and dynamics of microbial communities in various conditions and ecological habitats related to environmental and industrial biotechnological processes as well as on the microbial interactions by using molecular and classic methods. In the first part of the present thesis it was evaluated the efficiency of the Mediterranean biomixture (the main part of a biobed) in the degradation of fungicides generally used to control pests in vineyards and to assess their effects on microbial community. Biobeds are biological systems developed all over EU countries to protect water-bodies from pesticide contamination at farm level. Results showed that the biomixture had a good capability of degrading pesticides. Indeed, at the end of the experiment (112 days), the concentration of most of the pesticides was close to complete degradation. Denaturing gradient gel electrophoresis (DGGE) analysis showed an evident modification of microbial diversity after the addition of fungicides. However, at the end of degradation process, no significant changes in the composition of microbial community were seen. In the specific substrate used in the biomixture, both yeast flora and ascomycete filamentous fungi seemed to be involved in the degradation activity. In the second part of this PhD thesis it was investigated on the possible use of crude glycerol as a soil amendment. Crude glycerol is a principal by-product of biodiesel production. As the biodiesel industry is rapidly expanding over the last years, a glut of crude glycerol is being created, so there is an urgent need to find alternative uses for this by-product. Considering that crude glycerol is rich in organic compounds, a laboratory experiment was carried out to study the effect of different glycerol amounts on microbial diversity and chemical and biochemical properties of agricultural soil in order to assess the possible agronomical use of crude glycerol without further purification. Results of culture dependent methods showed that the addition of crude glycerol has stimulated the growth of cultivable bacteria and fungi, while DGGE analysis demonstrated that the added glycerol increase bacterial diversity in the soil. Microbial activity in all treated soils reached a steady-state condition in terms of daily respiration activity indicating a positive adaptation of the microbial biomass. This initial investigation showed that the crude glycerol does not seem to have negative effects on the soil but rather causes the development of microbial communities and increases microbial diversity. In the third part of this thesis, the yeast communities colonising grape berry surfaces were evaluated for the influence of fungicide treatments in an organic vineyard (copper/sulphur-based products) and a conventional vineyard (commonly used fungicides). Analysis of yeast abundance and diversity was carried out on grape berries and the grape juice during fermentation, using culture-dependent and -independent approaches. Yeast abundance was as generally reported for mature grapes, with higher counts from grapes treated with conventional fungicides. Direct isolation and DGGE analysis of initial grape samples showed less yeast species diversity in the organic vineyard compared with the conventional vineyard. In the organic and conventional vineyards, the dominant yeasts were Candida zemplinina and Hanseniaspora uvarum, respectively, typical species that colonise surfaces of mature grape berries. Saccharomyces cerevisiae was isolated by traditional methods and detected by DGGE only at the end of fermentation, with lower levels in the organic samples. Moreover, S. cerevisiae showed less intraspecific diversity in the organic samples (two genotypes), in comparison with the conventional samples (six genotypes). Altogether, these results show that the copper and sulphur treatments have greater negative influences on abundance and diversity of grape berry yeast communities (included S. cerevisiae), in comparison with fungicides used for conventional treatments. In the fourth part of the thesis, the attention was focused on the yeast interactions during wine fermentation. The use of a multistarter fermentation process with S. cerevisiae and non Saccharomyces wine yeasts has been proposed to simulate natural must fermentation and to confer greater complexity and specificity to wine. The aim of this work was to study the influence of Starmerella bombicola on S. cerevisiae gene expression and enzymatic activity of pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1), the two key enzymes of the alcoholic fermentation pathway. The presence of S. bombicola immobilized cells in a mixed fermentation trial confirmed an increase in fermentation rate, a combined consumption of glucose and fructose, an increase in glycerol and a reduction in the production of ethanol as well as a modification in the fermentation byproducts. Also the alcoholic fermentation of S. cerevisiae was influenced by S. bombicola immobilized cells. Indeed, Pdc1 activity in mixed fermentation was lower than that exhibited in pure culture while Adh1 activity showed an opposite behavior. The expression of both PDC1 and ADH1 genes was highly induced at the initial phase of fermentation which was more evident in mixed culture. S. bombicola immobilized cells greatly affected the fermentation behavior of S. cerevisiae and the analytical composition of wine. The influence of S. bombicola was not limited to a simple additive contribution but its presence caused metabolic modifications during S. cerevisiae fermentation causing variation in the gene expression and enzymatic activity of alcohol deydrogenase and pyruvate decarboxylase.

La presenza di corpi apoptotici (chiamati anche M540 bodies a causa della loro colorazione con la merocianina 540) nell’eiaculato è stata ampiamente dimostrata. Tali M540 bodies sono elementi eterogenei sia per dimensioni che per densità e sono caratterizzati dalla scarsità di materiale cromatinico e dall’espressione di markers apoptotici, inclusi il recettore Fas e la frammentazione del DNA; sono maggiormente riscontrabili nell’eiaculato di uomini con scarsa qualità del liquido seminale. La presenza di M540 bodies nell’eiaculato ha sia implicazioni cliniche che metodologiche. Infatti, da un lato la loro presenza può essere considerata un segno di apoptosi patologica/eccessiva negli organi riproduttivi maschili; dall’altro, poiché tali corpi apoptotici mostrano dimensioni e densità simili a quelli della testa degli spermatozoi, possono rappresentare un fattore confondente negli studi di spermatozoi in citofluorimetria. Per questa ragione, abbiamo messo a punto una strategia di analisi off-line in modo da poter distinguere tali corpi apoptotici in base all’amplificazione del segnale di fluorescenza relativo alla colorazione nucleare; infatti gli M540 bodies hanno un basso contenuto di DNA, inferiore a quello degli spermatozoi. Perciò l’analisi off-line permette di discriminare i corpi apoptotici dai gameti maschili in base alle dimensioni e al contenuto di DNA, evitando in tal modo dati equivoci. Un ulteriore scopo di tale lavoro di tesi è stato quello di analizzare la presenza dei corpi apoptotici nei campioni seminali di uomini fertili appartenenti a quattro differenti popolazioni (da Groenlandia, Svezia, Polonia e Ucraina) ed in particolar modo di investigare se esistesse una correlazione tra la presenza di M540 bodies e l’esposizione ad organoclorurati biopersistenti, che possono agire come interferenti endocrini. In particolare sono stati presi in considerazione il CB-153 per la sua stretta correlazione con la concentrazione dei PCB totali, e il DDE (il principale metabolita del DDT) in quanto buon indicatore dell’esposizione allo stesso DDT. Inoltre è stato analizzato l’impatto del fumo di sigaretta sulla qualità seminale, sul danno al DNA degli spermatozoi e sulla presenza dei corpi apoptotici nell’eiaculato umano. E’ stata infine verificata l’esistenza di un possibile effetto epigenetico, prendendo in considerazione l’assunzione di fumo da parte delle madri degli uomini coinvolti nel progetto.