Effects of Semichronic Ethanol Ingestion on Functional Sperm Parameters and Chromatin Decondensation After in vitro Fertilization in outbred mouse

SANCHEZ M; FONTANA V; CALVO L; CALVO JC; CEBRAL E

Simposio; Symposium in Frontiers in Reproduction (FIR; 2016

Many experimental animal modelsdemonstrate that male alcohol consumption produces deleterious effectsdepending on concentration, time of exposure, form of ethanol administrationand blood alcohol levels (BAL). Chronic ethanol consumption impairs malereproductive function through its action on the hypothalamic-pituitary-gonadalaxis. At central level, ethanol inhibits LHRH release, thus decreasing LHpulses. At gonadal level, it induces testicular damage; inhibits testosteroneproduction, alters Sertoli cells function, induces germ cell apoptosis andproduces sperm abnormal morphology. To date, there is not enough informationabout the impact of semichronic consumption of alcohol on sperm parameters. Theaim of the present study is to analyze the effects of short term ingestion ofmoderate concentration of ethanol on morphological and functional parameters offertility evaluating together the effects on sperm chromatin decondensation. Toperform the experiments, CF-1 male mice (60 days old) were treated with ethanol15% in drinking water for 2 weeks (BAL: 20-65 mg/dL). At the same time, controlanimals received water.  Sperm wererecovered from epididymis and concentration, motility and viability(Hypo-osmotic swelling test, HOS) was measured as well as their morphology(Giemsa stain), sperm capacitation (Hyperactivation) and spontaneous andprogesterone-induced acrosomal reaction (HOS-SPERMAC). The in vitro decondensation of the spermatozoa was performed in thepresence of glutathione (10 mmol/ L) and heparin (4.6 µM) for 30 and 60 minutes. Inaddition, in vitro fertilization(IVF) was evaluated at different post-insemination times (2.5 (T1), 3.5 (T2)and 4.5 (T3) hours), after coincubating oocytes from CF-1 control mice withsperm from control (C) or treated CF-1 mice. Sperm from ethanol-treated mice had increasedsperm percentage with abnormal head (Control = 6.9 ± 0.1 versustreated= 10.5 ± 1.5 (n= 9), p<0.05, Student´s t-Test) and with abnormal flagellum(C = 8.9 ± 0.8 versus treated= 16.8 ± 2.1 (n= 9), p<0.05, Student´s t-Test). The hyperactivated sperm % was lower intreated mice than the controls, at 60 (C = 19.4 ± 2.8 versus treated= 10.6 ± 2.2 (n=6),p<0.05, Student´s t-Test) and 120 minutes of capacitation (C = 23.7 ± 2. 6 versus treated = 14.1 ± 2.5(n=6), p<0.05, Student´s t-Test). Acrosome reaction decreased in treated micecompared to control mice, determined at 120 (C = 32.9 ± 6.1 versus treated = 17.9 ± 2.5(n= 6), p<0.05) and 150 minutes of capacitation (C = 23.7 ± 2.6 versus treated = 14.1 ± 2.5(n= 6), p<0.01). In vitro sperm chromatindecondensation percentage was higher in treated mice when compared to controls (C = 48.3 ± 3.2versus treated = 57.0 ± 3.6 (n= 8), p<0.05 Student´s t-Test). The analysis of timecourse of in vitro fertilization showed a significant increasedfertilized oocyte % with decondensed sperm heads in treated mice compared tothe control in T1 (71.7 % vs 45.7%, p<0.05, Fisher?s Test), oocyte % with 2 pronucleusin T2 (82.0% vs 60.0%, p<0.05) and T3 (80.5% vs 63.2%, p<0.05) while the total % offertilized oocyte at T1 and T3 in ethanol-treated group was significantly highercompared to % of the control. Despite the lower blood ethanol level obtained,semichronic moderate ethanol exposure induced morphological and functionaldisorders in epididymal spermatozoa as well as alterations in IVF dynamics.These results suggest that the short term ethanol consumption, in outbredmouse, induces oligo-astenozoospermia and teratozoospermia leading toalterations in male fertility and zygote formation by a deregulated timing ofIVF.