Flow cytometry (FCM) has developed into a highly reliable technology that can serve the andrology laboratory and infertility practices. New fluorescent stains and techniques that have potential application to the FCM evaluation of sperm DNA\chromatin integrity have continuously being developed taking into consideration the pivotal role of sperm genome and epigenome for successful reproduction and embryo development. All these assays rely on DNA staining for the correct discrimination of sperms from the other cellular components of ejaculates, but the precise determination of sperm DNA content (C-value) has been hindered by the peculiar sperm’s flattened shape and by the tightly packed nature of DNA in sperm chromatin. In this study we set up a protocol of sperm head in vitro controlled decondensation that eliminates the troublesome effect of the variable degree of chromatin condensation between different sperm cells, ensuring the stoichiometric staining of sperm DNA and the proper gating of aploid sperms needed for any additional FCM analysis. Washed sperm samples from volunteers of our Department were fixed in a stabilization solution containing aldehyde-fixative. After washing they were incubated at 25°C for 30’ in a decondensation solution containing 1,4-Dithiothreitol (DTT), Heparin and Triton. Decondensed sperms were postfixed in ethanol 70%, and re-suspended in PBS/BSA. DNA staining was performed for 30’ at RT in 0.5 mL Propidium Iodide (P.I.)/RNase staining solution (40 μg P.I. and 0.1 mg RNase/DNase-free) before FCM analysis. Chicken Red Blood Cells (CRBC), were used as stable c-value Internal Standard. FCM was performed by an Epics XL Flow Cytometer (Beckman Coulter-IL, USA). Histograms of sperm DNA content value distribution were used to quantify the degree of sperm DNA accessibility to P.I. and to others DNA staining dyes, before, during and after the decondensation. All offline analysis were done on FCS files using Weasel or WinMDI software. Fluorescence Intensity (FI) of samples is expressed as Mean Fluorescence Channel (MFC) and the genome size (picograms of DNA per cell nucleus) was calculated by considering the CRBC and human diploid cells c-values. Native nondecondensed fluorochrome-labeled sperms showed a significant under-staining resulting in FI emission corresponding to a C-value variable around 1,4 pg for haploid cells. Distributions of FI after our sperm head decondensation protocol proved an increase of MFC of the sample in accordance to the correct stoichiometric staining of sperm DNA (haploid human C-value 3.56 pg). Peaks of decondensed sperm histograms are clearly associated with haploid DNA Content and the stoichiometric staining of DNA allows the direct evaluation of sperm DNA Content and diploid sperm population which peak overlaps the expected diploid DNA content of human species. Both DTT and Heparin at specific concentration were required for an efficient decondensation procedure with no induced DNA loss or significant increase in sperm DNA fragmentation (analysed by microspectrophotometric DNA quantification and TUNEL assays). Physiological chromatin compaction of mature sperms results in chemical restricted accessibility of DNA, therefore staining of sperm nuclei by DNA-specific dyes is not stoichiometric during most of the stages of sperm maturation. Conversely, the highly variable distribution of DNA staining of sperm nuclei becomes rather uniform after our sperm chromatin decondensation procedure. The main peaks of C-value histograms exactly overlaps the expected haploid DNA content of human sperm. This novel sperm head decondensation protocol will found a series of applications on the study of sperm samples by FCM. For istance, the stoichiometric staining of sperm DNA allows the estimation of the total chromosomes sperm aneuploidy frequency of semen samples by the C-value distribution profile analysis and the direct quantification of diploid sperm number. Moreover, it ensures the proper DNA accessibility and cell integrity to make applicable the Sperm-Flow-FISH assay for a rapid and accurate estimation of chromosome specific aneuploidy sperm rate
La Citofluorimetria a flusso (FCM) è una metodica altamente affidabile con enormi applicazioni nello studio dei campioni biologici. Le grandi opportunità offerte da questa tecnica sono state fatte proprie anche dai laboratori di andrologia per lo studio del campione seminale. Nuove metodiche vengono continuamente sviluppate, soprattutto per la valutazione dell’integrità del DNA/cromatina delle cellule nemaspermiche. La peculiare forma appiattita della testa e l’alto grado di condensazione del materiale genetico degli spermatozoi sono i principali problemi dello studio citofluorimetrico del liquido seminale. In questo studio è stato messo a punto un nuovo protocollo di decondensazione del DNA degli spermatozoi in vitro che elimina il problema dovuto alla compattazione del DNA, assicurando una colorazione stechiometrica del DNA stesso. I campioni seminale sono stati lavati e fissati in una soluzione di stabilizzazione contenente un fissativo aldeidico. Dopo rimozione del fissativo, sono stati incubati a 25°C per 30’ in una soluzione di decondensazione contenente 1,4-Ditiotreitolo (DTT), eparina, e TritonX100. Gli spermatozoi decondensati sono stati post-fissati in etanolo 70% e risospesi in PBS-BSA per la colorazione del DNA, eseguita per 30’ a R.T. in una soluzione contenente ioduro di propidio (P.I.) e RNasi (40 μg P.I. and 0.1 mg RNase/DNase-free). Eritrociti di pollo (CRBC) sono stati usati come standard interno per la valutazione del C-value. L’analisi FCM è stata eseguita con un Epics XL Flow Cytometer . Gli istogrammi di distribuzione del valore di DNA-Content sono stati usati per quantificare il grado di accessibilità del DNA degli spermatozoi al P.I. prima, durante e dopo la decondensazione. Le analisi off-line sono state eseguite su files FCS usando i software Weasel o WinMDI. L’intensità di fluorescenza (FI) dei campioni analizzati è espressa come canale di fluorescenza medio (MFC) e il genome size (picogrammi di DNA/nucleo) è stato calcolato considerando i C-value dei CRBC e di cellule diploidi umane. Gli spermatozoi non decondensati colorati con PI mostrano una significativa sottocolorazione in termini di FI, corrispondente a un C-value di 1,4 pg. Dopo la procedura di stabilizzazione e decondensazione, le distribuzioni di FI mostrano un aumento dell’MFC del campione corrispondente a un C-value di 3,56 pg, tipico delle cellule umane aploidi. I picchi degli istogrammi dei campioni seminali de condensati corrispondono perfettamente ad un contenuto di DNA aploide e la colorazione stechiometrica del DNA permette la valutazione diretta del contenuto di DNA degli spermatozoi e della frequenza di spermatozoi diploidi i cui picchi si sovrappongono con quelli relativi a cellule umani diploidi. Un’efficiente procedura di decondensazione, senza perdita di DNA o aumento del grado di frammentazione indotta, richiede specifiche concentrazioni di DTT ed eparina. La fisiologica compattazione della cromatina degli spermatozoi maturi limita l’accessibilità al DNA rendendo impossibile una colorazione stechiometrica da parte di coloranti DNA-specifici durante la maggior parte degli stadi finali della spermiogenesi. Il nostro protocollo di decondensazione in vitro permette una colorazione uniforme e stechiometrica del DNA, candidandosi come metodo di elezione per una serie di applicazioni nello studio del campione seminale in FCM. Ad esempio la colorazione stechiometrica del DNA permette una stima delle frequenza di aneuploidie e la diretta quantificazione della percentuale di spermatozoi diploidi.
Messa a punto di un nuovo protocollo di decondensazione del DNA degli spermatozoi in vitro, e applicazioni in citofluorimetria a flusso
ANTONUCCI, NIKI
2011
Abstract
Flow cytometry (FCM) has developed into a highly reliable technology that can serve the andrology laboratory and infertility practices. New fluorescent stains and techniques that have potential application to the FCM evaluation of sperm DNA\chromatin integrity have continuously being developed taking into consideration the pivotal role of sperm genome and epigenome for successful reproduction and embryo development. All these assays rely on DNA staining for the correct discrimination of sperms from the other cellular components of ejaculates, but the precise determination of sperm DNA content (C-value) has been hindered by the peculiar sperm’s flattened shape and by the tightly packed nature of DNA in sperm chromatin. In this study we set up a protocol of sperm head in vitro controlled decondensation that eliminates the troublesome effect of the variable degree of chromatin condensation between different sperm cells, ensuring the stoichiometric staining of sperm DNA and the proper gating of aploid sperms needed for any additional FCM analysis. Washed sperm samples from volunteers of our Department were fixed in a stabilization solution containing aldehyde-fixative. After washing they were incubated at 25°C for 30’ in a decondensation solution containing 1,4-Dithiothreitol (DTT), Heparin and Triton. Decondensed sperms were postfixed in ethanol 70%, and re-suspended in PBS/BSA. DNA staining was performed for 30’ at RT in 0.5 mL Propidium Iodide (P.I.)/RNase staining solution (40 μg P.I. and 0.1 mg RNase/DNase-free) before FCM analysis. Chicken Red Blood Cells (CRBC), were used as stable c-value Internal Standard. FCM was performed by an Epics XL Flow Cytometer (Beckman Coulter-IL, USA). Histograms of sperm DNA content value distribution were used to quantify the degree of sperm DNA accessibility to P.I. and to others DNA staining dyes, before, during and after the decondensation. All offline analysis were done on FCS files using Weasel or WinMDI software. Fluorescence Intensity (FI) of samples is expressed as Mean Fluorescence Channel (MFC) and the genome size (picograms of DNA per cell nucleus) was calculated by considering the CRBC and human diploid cells c-values. Native nondecondensed fluorochrome-labeled sperms showed a significant under-staining resulting in FI emission corresponding to a C-value variable around 1,4 pg for haploid cells. Distributions of FI after our sperm head decondensation protocol proved an increase of MFC of the sample in accordance to the correct stoichiometric staining of sperm DNA (haploid human C-value 3.56 pg). Peaks of decondensed sperm histograms are clearly associated with haploid DNA Content and the stoichiometric staining of DNA allows the direct evaluation of sperm DNA Content and diploid sperm population which peak overlaps the expected diploid DNA content of human species. Both DTT and Heparin at specific concentration were required for an efficient decondensation procedure with no induced DNA loss or significant increase in sperm DNA fragmentation (analysed by microspectrophotometric DNA quantification and TUNEL assays). Physiological chromatin compaction of mature sperms results in chemical restricted accessibility of DNA, therefore staining of sperm nuclei by DNA-specific dyes is not stoichiometric during most of the stages of sperm maturation. Conversely, the highly variable distribution of DNA staining of sperm nuclei becomes rather uniform after our sperm chromatin decondensation procedure. The main peaks of C-value histograms exactly overlaps the expected haploid DNA content of human sperm. This novel sperm head decondensation protocol will found a series of applications on the study of sperm samples by FCM. For istance, the stoichiometric staining of sperm DNA allows the estimation of the total chromosomes sperm aneuploidy frequency of semen samples by the C-value distribution profile analysis and the direct quantification of diploid sperm number. Moreover, it ensures the proper DNA accessibility and cell integrity to make applicable the Sperm-Flow-FISH assay for a rapid and accurate estimation of chromosome specific aneuploidy sperm rateFile | Dimensione | Formato | |
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Tesi.Antonucci.pdf
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0. CONTENTS.pdf
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1. INTRODUCTION.pdf
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2. SET UP OF A NOVEL IN-VITRO SPERM HEAD DECONDENSATION PROTOCOL FOR FLOW CYTOMETRIC ANALYSIS.pdf
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3.1 APPLICATIONS OF THE NOVEL IN-VITRO SPERM HEAD DECONDENSATION PROTOCOL..pdf
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3.2 FLOW CYTOMETER DETERMINATION OF DIPLOIDS.pdf
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3.3 FLOW CYTOMETER DETERMINATION OF HUMAN SPERM DNA FRAGMENTATION.pdf
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4. FINAL DISCUSSION.pdf
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5. PUBLICATIONS.pdf
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https://hdl.handle.net/20.500.14242/97997
URN:NBN:IT:UNIVPM-97997