Techniques for sperm evaluation using fluorescent probes

Abstract

A variety of laboratory tests were developed to obtain more reliable results of sperm evaluation and increase the accuracy of sperm fertility predictions. These tests detected damage of sperm specific compartments or organelles, which cannot be detected in routine sperm analysis. The use of fluorescent probes and detection using fluorescent microscopy or flow cytometry is an important tool but a more precise and accurate laboratory test is needed. Propidium iodide and 6-carboxyfluorescein diacetate are used for evaluations of plasmatic membrane integrity. Fluorescein isothiocyanate, associated with conjugated lecithin Psium sativum or Arachis hypogaea, are used for evaluations of acrosome integrity. Two probes, MitoTracker or Rhodamine123, are generally used to measure the absence or presence of mitochondrial potential. However, a better option is 5,5'; 6,6' -tetrachloro -1,1'; 3,3' -tetraetilbenzimidazolil-carbocyanine (JC-1) dye, which assesses not only the presence of mitochondrial potential and distinguished spermatozoa with poorly and highly functional mitochondria. Two techniques, TUNEL or COMETA, and the Acridine Orange Test (AOT) dye are used to evaluate chromatin integrity. A fluorescence technique based on chlortetracycline (CTC) or Merocyanine 540 is used to estimate whether sperm pass by or through the capacitation process. This review focuses on the fluorescent probes that are most widely used to evaluate plasma membrane integrity, capacitation, acrosome integrity, chromatin integrity and mitochondrial potential. Key words: Fluorescence microscopy, sperm, cryopreservation ResumoDiversos testes laboratoriais têm sido desenvolvidos com o intuito de obter resultados confiáveis nas avaliações espermáticas, aumentando a qualidade e a confiabilidade do sêmen utilizado em técnicas de reprodução assistida. Esses testes têm permitido detectar danos em compartimentos e organelas específicas da célula espermática, que não são detectados nas análises de rotina. Dentre esses testes, o uso de sondas fluorescentes e sua detecção em microscópio de epifluorescência ou citometria de fluxo se tornaram ferramenta importante quando uma avaliação mais acurada é necessária. Para a avaliação da integridade de membrana plasmática, pode ser utilizado o iodeto de propídio, associado ao diacetato de 6-carboxifluoresceína. As avaliações de integridade acrossomal podem ser feitas através do isotiocianato de fluoresceína, associado às lecitinas conjugadas, como a Psium sativum ou Arachis hypogaea. O potencial mitocondrial pode ser avaliado quanto à ausência ou presença através da sonda Mitotracker ou Rodamina 123. Outra opção ainda melhor pode ser observada utilizando o corante iodeto de 5,5'; 6,6' -tetracloro -1,1'; 3,3' -tetraetilbenzimidazolil-carbocianina (JC-1), este corante avalia a presença do potencial mitocondrial e avalia quanto à classificação do grau. Para avaliar a integridade

Effects of Cryopreservation on Gene Expression and Post Thaw Sperm Quality of Pacific Abalone, Haliotis discus hannai

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Effects of Antifreeze Protein III on Sperm Cryopreservation of Pacific Abalone, Haliotis discus hannai

“…AI is a key indicator of fertility potential. It can be visualized using LYSO-G florescent dye [39]. In the present study, the highest percentage of intact acrosome was found for sperm cryopreserved using 10 µg/mL of AFPIII combined with 8% DMSO.…”

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“…According to Ravagnan et al (2002), after the rupture of mitochondrial membranes, there is a release of pro-apoptotic factors, which can cause irreparable structural and functional damage to sperm. Although the PI−CMXRos−Hoechst 342 combination was efficient for the analysis of cavies’ sperm, we emphasize that future studies should consider the use of other biomarkers, including those used for the analysis of specific regions of the sperm as the acrosome, chromatin and membranes, as well as those used for evaluating mitochondrial potential and sperm capacitation (Cunha et al , 2015).…”