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SHORT COMMUNICATIONProtective effect of fallopian tubal ﬂuid against activatedleucocyte-induced sperm DNA fragmentation: preliminaryresultsP. Navarrete Gomez1,6, J. Espinoza Ruiz1,5, J. Parodi Rivera1, J. G. Alvarez2,3 & R. Sanchez Gutierrez4 ´ ´ ´1 ´ ´ Centro de Excelencia de Biotecnologıa de la Reproduccion (CEBIOR), Universidad de La Frontera, Temuco, Chile;2 ` Instituto Marques, Barcelona, Barcelona, Spain;3 ´ ` Fundacion Leonardo Marques, Barcelona, Spain;4 ´ Departamento de Ciencias Preclınicas, Universidad de La Frontera, Temuco, Chile;5 Tesista y;6 ´ ´ Estudiante del Programa de Doctorado en Ciencias Mencion Biologıa Celular y Molecular Aplicada, Becarios CONICYTKeywords: SummaryActivated leucocytes—DNAfragmentation—human sperm—ROS— The integrity of the paternal genome is of paramount importance in the initia-tubal ﬂuid tion and maintenance of a viable pregnancy. Oxygen radicals (ROS) have been identiﬁed as one of the main factors responsible for the induction of spermCorrespondence DNA damage. Spermatozoa are mainly protected against ROS-induced damage ´ ´ ´Dr Raul Sanchez Gutierrez, Facultad de by seminal plasma. However, this protective effect disappears once spermatozoaMedicina, Universidad de La Frontera, ManuelMontt 112, Temuco, Chile. enter the female genital tract. The fallopian tube mucosa may play a protectiveTel.: +56 45 744248; role against ROS-induced sperm damage. The main objective of this study wasFax: +56 45 325600; to determine whether human tubal explants and tubal ﬂuid exert a protectiveE-mail: firstname.lastname@example.org effect on ROS-induced sperm DNA damage. Spermatozoa were exposed to tubal explants and/or tubal ﬂuid in the presence of phorbol myristate acetateAccepted: January 13, 2009 (PMA)-activated polymorphonuclear leucocytes or control medium and sperm DNA fragmentation was measured using the TdT-mediated dUTP-biotin nick end labelling (TUNEL) test. Exposure of human spermatozoa to PMA-activated leucocytes resulted in a 2-fold increase in sperm DNA fragmentation. Co-incu- bation of spermatozoa with tubal explants did not reduce this damage. However, pre-incubation of spermatozoa with tubal ﬂuid resulted in a statis- tically signiﬁcant reduction in sperm DNA fragmentation levels, comparable to those observed in control. In conclusion, tubal ﬂuid appears to protect against activated leucocyte-induced sperm DNA fragmentation, thus preserving the integrity of the paternal genome. ﬂuid exert a protective effect against ROS-induced spermIntroduction DNA damage.The integrity of the paternal genome is vital to the onsetand maintenance of a viable pregnancy to term Materials and methods(Angelopoulou et al., 2007). The previous studies haveshown that exposure of human spermatozoa to tubal/ Tubal explants were obtained from the fallopian tubes ofoviductal epithelial cells protects them from ROS-induced four healthy women subjected to hysterectomy. Tubaldamage, thus increasing sperm survival (El Mouatassim ﬂuid was obtained from the explants and incubated atet al., 2000). Despite all these studies, however, it is still 37 °C for 24 h in supplemented DMEM mediumnot known whether the oviduct exerts a protective effect (sDMEM). Semen samples were obtained from healthyon sperm DNA fragmentation. The primary aim of this donors, processed for standard swim-up and incubatedstudy was to determine if human tubal explants or tubal with tubal ﬂuid during 12 h.196 ª 2009 Blackwell Verlag GmbH Æ Andrologia 41, 196–198
´P. Navarrete Gomez et al. Tubal ﬂuid protects against sperm DNA damage Polymorphonuclear (PMN) leucocytes were activated (a)with phorbol-12-miristate-13-acetate (PMA). Sperm DNAfragmentation was determined using the TUNEL testaccording to the manufacturer’s instructions (Roche,Grenzach-Wyhlen, Germany). Following TUNEL labelling,propidium iodide was added and the samples analysed byﬂow cytometry. TUNEL test data were analysed using thegraphpad prism 5.0 program (GraphPad Software, LaJolla, CA, USA). Differences between the groups were analysed using theKruskal–Wallis and Dunn’s post-tests. A P-value less than0.05 (P < 0.05) was considered as statistically signiﬁcant. (b)Results and discussionIncubation of human spermatozoa in sDMEM did notresult in any signiﬁcant increase in DNA fragmentationcompared with the control with HTF medium (P > 0.05)(Fig. 1a). Treatment with DNAse I produced a fragmenta-tion level of 95 ± 3.06% (P < 0.05). Incubation of humanspermatozoa with activated leucocytes resulted in anincrease in DNA fragmentation levels, although these dif-ferences were not statistically signiﬁcant (P > 0.05).Moreover, incubation of the spermatozoa with PMN,PMA and DMSO did not produce any signiﬁcant effect (c)(P > 0.05) on sperm DNA fragmentation. Incubation ofhuman spermatozoa with tubal explants produced a sig-niﬁcant decrease in sperm DNA fragmentation levels dur-ing incubation at 37 °C (P < 0.05) (Fig. 1b), whereasincubation of human spermatozoa with tubal mediumdid not result in any signiﬁcant effect on sperm DNAfragmentation (P > 0.05). Incubation with activated leu-cocytes did not produce any signiﬁcant decrease(P > 0.05) in activated leucocyte-induced sperm DNAfragmentation levels, whereas incubation with tubal ﬂuid,after incubation with activated leucocytes, produced a sig-niﬁcant decrease in sperm DNA fragmentation levels(P < 0.05) comparable to those observed in the control(Fig. 1c). Fig. 1 Effect of incubation in sDMEM, tubal explants (TU) or tubal The main ﬁndings of this study are that (i) tubal ex- ﬂuid (CM) on sperm DNA fragmentation. (a) Percentage of TUNEL-plants decrease sperm DNA fragmentation during incuba- positive spermatozoa after incubation in sDMEM (*P < 0.05); (b) Per-tion at 37 °C, whereas tubal ﬂuid had no effect; and (ii) centage of TUNEL-positive spermatozoa after incubation with TU ortubal ﬂuid prevents sperm DNA fragmentation induced CM; (c) Percentage of TUNEL-positive spermatozoa after incubationby PMA-activated leucocytes, whereas tubal explants had with tubal TU or CM prior to incubation with activated leucocytes andno effect. The previous studies have shown that human their respective controls.spermatozoa can establish direct contact with the tubalepithelium and that this contact increases motility, pre- effect on activated leucocyte-induced sperm DNA frag-vents the premature acrosome reaction and increases the mentation might be related to tubal cell damage inducedfertility capacity of spermatozoa (Morales et al., 1996; by the speciﬁc ROS produced by activated leucocytes,Green et al., 2001). This could be related to a possible which could inactivate enzymatic antioxidant defences ofantioxidant effect of the tubal cells that would neutralise these cells. In the case of tubal ﬂuid, the antioxidantthe ROS produced by spermatozoa in the extracellular defences would be mainly nonenzymatic soluble factorsmedium. In contrast, the tubal explants’ lack of protective that are not susceptible to inactivation by the high levelsª 2009 Blackwell Verlag GmbH Æ Andrologia 41, 196–198 197
Tubal ﬂuid protects against sperm DNA damage ´ P. Navarrete Gomez et al.of ROS produced by the activated leucocytes. Studies are ´ ´ ´ El Mouatassim S, Guerin P, Menezo Y (2000) Mammalianunderway to try to answer the question of why the tubal oviduct and protection against free oxygen radicals:ﬂuid protects against activated leucocyte-induced DNA expression of genes encoding antioxidant enzymes indamage and not that produced during incubation with human and mouse. Eur J Obstet Gynecol Reprod Biol 89:nonactivated leucocytes. 1–6. Green CE, Bredl J, Holt WV, Watson PF, Fazeli A (2001) Carbohydrate mediation of boar sperm binding to oviductalReferences epithelial cells in vitro. Reproduction 122:305–315. ´ Morales P, Palma V, Salgado AM, Villalon M (1996) SpermAngelopoulou A, Plastira K, Msaouel P (2007) Spermatozoal sensitive biomarkers to defective protaminosis and frag- interaction with human oviductal cells in vitro. Hum Reprod mented DNA. Reprod Biol Endocrinol 5:1–15. 11:1504–1509.198 ª 2009 Blackwell Verlag GmbH Æ Andrologia 41, 196–198