265 TESTICULAR DEGENERATION AFFECTED PLASMA, ACROSOMAL AND MITOCHONDRIAL MEMBRANE INTEGRITY, AND DNA FRAGMENTATION IN RAM SPERMATOZOA

2015 ◽  
Vol 27 (1) ◽  
pp. 222
Author(s):  
M. Bianchi Rodrigues Alves ◽  
A. Furugen Cesar de Andrade ◽  
R. Paes de Arruda ◽  
L. Batissaco ◽  
R. Lançoni ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Potential ◽  
Dna Fragmentation ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Membrane Integrity ◽  
Mitochondrial Potential ◽  
Plasma Membrane Integrity ◽  
Scrotal Insulation ◽  
Testicular Degeneration

Testicular degeneration, an important cause of male infertility, adversely affects sperm motility and morphology. However, few studies describe effects on integrity of plasma and acrosomal membranes, mitochondrial membrane potential, and DNA fragmentation; therefore, they were evaluated in the present study. Testicular degeneration was induced in 17 White Dorper rams (scrotal insulation for 72 h). Semen was collected (artificial vagina) twice before insulation and twice thereafter (15-day intervals between post-insulation collections). Sperm motility and morphology were analysed by SCA software (Sperm Class Analyser®, MICROPTIC®, Barcelona, Spain) and differential interference contrast microscopy (DIC, model 80i, Nikon, Tokyo, Japan), respectively. Membrane integrity and potential were assessed with fluorescent probes: Hoescht 33342, propidium iodide, FITC-PSA, and JC-1 (Celeghini et al. 2010 Arq. Bras. Med. Vet. Zootec. 62, 536–543) and imaged with fluorescence microscopy (Nikon Model 80i, Nikon, Tokyo, Japan). Fragmentation of DNA was evaluated with a Halomax® kit (Halotech® DNA, Madrid, Spain). Data were analysed with Statview software (Stat View 1998, SAS Institute Inc., Cary, NC, USA). Data obtained from the periods (before × after insulation) were evaluated by analysis of variance (ANOVA) and means were compared using Tukey's test. Total motility (before: 87.53 ± 1.21%; after: 46.53 ± 4.46%) and progressive motility (before: 58.64 ± 2.00%; after: 31.33 ± 3.82%) were reduced (P < 0.01) by scrotal insulation, as were sperm major defects (before: 10.64 ± 1.65%; after: 54.30 ± 3.67%) and total defects (before: 20.50 ± 2.40%; after: 63.85 ± 3.41%; P < 0.0001). Sperm with intact plasma and acrosomal membranes and high mitochondrial potential (PIAIH) decreased (P < 0.0001) after insulation. In that regard, 53.19 ± 2.20 and 28.48 ± 3.48% of sperm were classified as PIAIH before v. after insulation, respectively. Furthermore, plasma membrane integrity, acrosome membrane integrity, and high mitochondrial potential were assessed independently. The quantity of plasma membrane integrity cells (before: 62.01 ± 2.07%; after: 33.92 ± 3.94%), acrosome membrane integrity cells (before: 57.17 ± 2.30%; after: 31.47 ± 3.77%), and high mitochondrial potential cells (before: 85.72 ± 1.42%; after: 57.28 ± 3.12%) were also reduced (P < 0.0001) after insulation. Likewise, DNA integrity decreased (P = 0.002) from 98.87 ± 0.26% before insulation to 91.88 ± 2.6% afterward. In conclusion, sperm plasma and acrosomal membrane integrity, mitochondrial membrane potential, and DNA fragmentation were adversely affected by testicular degeneration in rams induced by scrotal insulation.Research was supported by FAPESP process 2012/00040-0 and 2011/16744-3.

scholarly journals Simultaneous evaluation of plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential in bovine spermatozoa by flow cytometry

Zygote ◽  
2015 ◽  
Vol 24 (4) ◽  
pp. 529-536 ◽  
Author(s):  
Chihiro Kanno ◽  
Sung-Sik Kang ◽  
Yasuyuki Kitade ◽  
Yojiro Yanagawa ◽  
Yoshiyuki Takahashi ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Plasma Membrane ◽  
Membrane Potential ◽  
Fluorescence Microscopy ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Membrane Integrity ◽  
Plasma Membrane Integrity ◽  
Bovine Spermatozoa ◽  
Acrosomal Integrity

SummaryThe present study aimed to develop an objective evaluation procedure to estimate the plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential of bull spermatozoa simultaneously by flow cytometry. Firstly, we used frozen–thawed semen mixed with 0, 25, 50, 75 or 100% dead spermatozoa. Semen was stained using three staining solutions: SYBR-14, propidium iodide (PI), and phycoerythrin-conjugated peanut agglutinin (PE–PNA), for the evaluation of plasma membrane integrity and acrosomal integrity. Then, characteristics evaluated by flow cytometry and by fluorescence microscopy were compared. Characteristics of spermatozoa (viability and acrosomal integrity) evaluated by flow cytometry and by fluorescence microscopy were found to be similar. Secondly, we attempted to evaluate the plasma membrane integrity, acrosomal integrity, and also mitochondrial membrane potential of spermatozoa by flow cytometry using conventional staining with three dyes (SYBR-14, PI, and PE–PNA) combined with MitoTracker Deep Red (MTDR) staining (quadruple staining). The spermatozoon characteristics evaluated by flow cytometry using quadruple staining were then compared with those of staining using SYBR-14, PI, and PE–PNA and staining using SYBR-14 and MTDR. There were no significant differences in all characteristics (viability, acrosomal integrity, and mitochondrial membrane potential) evaluated by quadruple staining and the other procedures. In conclusion, quadruple staining using SYBR-14, PI, PE–PNA, and MTDR for flow cytometry can be used to evaluate the plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential of bovine spermatozoa simultaneously.

scholarly journals Integrity of the plasma membrane, the acrosomal membrane, and the mitochondrial membrane potential of sperm in Nelore bulls from puberty to sexual maturity

2016 ◽  
Vol 68 (3) ◽  
pp. 620-628 ◽  
Author(s):  
L.S.L.S. Reis ◽  
A.A. Ramos ◽  
A.S. Camargos ◽  
E. Oba
Keyword(s):  
Plasma Membrane ◽  
Mitochondrial Membrane ◽  
Sexual Maturity ◽  
Wave Motion ◽  
Membrane Integrity ◽  
Mitochondrial Potential ◽  
Plasma Membrane Integrity ◽  
Scrotal Circumference ◽  
Acrosomal Membrane ◽  
Sperm Membrane

ABSTRACT This study evaluated the plasma membrane integrity, acrosomal membrane integrity, and mitochondrial membrane potential of Nelore bull sperm from early puberty to early sexual maturity and their associations with sperm motility and vigor, the mass motility of the spermatozoa (wave motion), scrotal circumference, and testosterone. Sixty Nelore bulls aged 18 to 19 months were divided into four lots (n=15 bulls/lot) and evaluated over 280 days. Semen samples, collected every 56 days by electroejaculation, were evaluated soon after collection for motility, vigor and wave motion under an optical microscope. Sperm membrane integrity, acrosomal integrity, and mitochondrial activity were evaluated under a fluorescent microscope using probe association (FITC-PSA, PI, JC-1, H342). The sperm were classified into eight integrity categories depending on whether they exhibited intact or damaged membranes, an intact or damaged acrosomal membrane, and high or low mitochondrial potential. The results show that bulls have a low amount of sperm with intact membranes at puberty, and the sperm show low motility, vigor, and wave motion; however, in bulls at early sexual maturity, the integrity of the sperm membrane increased significantly. The rate of sperm membrane damage was negatively correlated with motility, vigor, wave motion, and testosterone in the bulls, and a positive correlation existed between sperm plasma membrane integrity and scrotal circumference. The integrity of the acrosomal membrane was not influenced by puberty. During puberty and into early sexual maturity, bulls show low sperm mitochondrial potential, but when bulls reached sexual maturity, high membrane integrity with high mitochondrial potential was evident.

scholarly journals Energy transduction in intact synaptosomes. Influence of plasma-membrane depolarization on the respiration and membrane potential of internal mitochondria determined in situ

1980 ◽  
Vol 186 (1) ◽  
pp. 21-33 ◽  
Author(s):  
I D Scott ◽  
D G Nicholls
Keyword(s):  
Plasma Membrane ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Significant Proportion ◽  
Membrane Potentials ◽  
Simultaneous Estimation ◽  
Anaerobic Glycolysis ◽  
Mitochondrial Potential ◽  
Plasma Membrane Potential

A method is described, based on the differential accumulation of Rb+ and methyltriphenylphosphonium, for the simultaneous estimation of the membrane potentials across the plasma membrane of isolated nerve endings (synaptosomes), and across the inner membrane of mitochondria within the synaptosomal cytoplasm. These determinations, together with measurements of respiratory rates, and ATP and phosphocreatine concentrations, are used to define the bioenergetic behaviour of isolated synaptosomes under a variety of conditions. Under control conditions, in the presence of glucose, the plasma and mitochondrial membrane potentials are respectively 45 and 148mV. Addition of a proton translocator induces a 5-fold increase in respiration, and abolishes the mitochondrial membrane potential. The addition of rotenone to inhibit respiration does not affect the plasma membrane potential, and only lowers the mitochondrial membrane potential to 128mV. Evidence is presented that ATP synthesis by anaerobic glycolysis is sufficient under these conditions to maintain ATP-dependent processes, including the reversal of the mitochondrial ATP synthetase. Addition of oligomycin under non-respiring conditions leads to a complete collapse of the mitochondrial potential. Even under control conditions the plasma membrane (Na+ + K+)-dependent ATPase is responsible for a significant proportion of the synaptosomal ATP turnover. Veratridine greatly increases respiration, and depolarizes the plasma membrane, but only slightly lowers the mitochondrial membrane potential. High K+ and ouabain also lower the plasma membrane potential without decreasing the mitochondrial membrane potential. In non-respiring synaptosomes, anaerobic glycolysis is incapable of maintaining cytosolic ATP during the increased turnover induced by veratridine, and the mitochondrial membrane potential collapses. It is concluded that the internal mitochondria must be considered in any study of synaptosomal transport.

scholarly journals Antisperm antibodies and sperm function in bulls undergoing scrotal insulation

Reproduction ◽  
2020 ◽  
Vol 160 (5) ◽  
pp. 783-792
Author(s):  
Maria S Ferrer ◽  
Roberto Palomares ◽  
David Hurley ◽  
Anna-Claire Bullington ◽  
Alejandro Hoyos-Jaramillo ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Membrane Integrity ◽  
Sperm Function ◽  
Antisperm Antibodies ◽  
Scrotal Circumference ◽  
Acrosome Integrity ◽  
Scrotal Insulation

Bovine antisperm antibodies (ASAs) have been associated with teratospermia and asthenospermia. It was hypothesized here that scrotal insulation induces the formation of ASAs and deterioration of sperm function. Scrotal insulation bags were placed in 10 bulls for 8 days. Semen was collected on days −29, −22 and −2, twice weekly from days 5 to 54, and thereafter weekly until day 96 (day 0 = first day of scrotal insulation). On each collection day, scrotal circumference, sperm motility, morphology, membrane integrity, acrosome integrity, apoptosis, lipid peroxidation, mitochondrial membrane potential, ASA binding and DNA integrity were evaluated. The percentage of IgG- and IgA-bound sperm increased between days 12 and 96 (P < 0.0001), in association with poor motility (days 19–30, P < 0.005) and morphology (days 8–40, P < 0.0001). Mean scrotal circumference decreased between days 15 and 75 (P < 0.0001). There was also a deterioration in sperm membrane integrity (days 19–40, P < 0.0001), acrosome integrity (days 26–89, P < 0.0001), lipid peroxidation (days 5–12, P < 0.0001), and mitochondrial membrane potential (days 12–96, P = 0.001). In contrast, a decrease in apoptotic cells (days 37–83, P = 0.0002) and lipid peroxidation (days 19–96, P < 0.0001) was noticed. Most bulls recovered normospermia by day 96. However, the persistence of ASAs, acrosomal damage and dysfunctional mitochondria suggest a long term effect of scrotal insulation on sperm function and the homeostasis of the reproductive immune system.

27 Effects of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone on frozen - thawed bovine sperm

2019 ◽  
Vol 31 (1) ◽  
pp. 139
Author(s):  
C. De Canditiis ◽  
N. Pagano ◽  
V. Longobardi ◽  
C. Zuchegna ◽  
M. A. Kosior ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Dna Fragmentation ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Membrane Integrity ◽  
Sperm Viability ◽  
Caspase Inhibitor ◽  
Osmotic Swelling ◽  
Bovine Sperm ◽  
Fluoromethyl Ketone

Semen cryopreservation is critical for appropriate planning of both AI and IVF trials, improving the benefit:cost ratio. However, cryopreservation induces damage in mammalian spermatozoa, resulting in decreased fertility (Medeiros et al. 2002 Theriogenology 57, 327-344). It is known that cryopreservation and thawing induce apoptosis in a variety of cells, including bovine sperm (Anzar et al. 2002 Biol. Reprod. 66, 354-360). Cryotolerance of in vitro-produced bovine embryos was recently improved by inhibiting apoptosis using a caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (ZVAD-FMK), during vitrification and subsequent culture (Pero et al. 2018 Theriogenology 108, 127-135). The aim of this work was to evaluate whether treatment of bovine frozen-thawed sperm with the caspase inhibitor Z-VAD-FMK may prevent aberrant apoptosis and consequently improve sperm viability. Six ejaculates from 12 bulls were used for the trial. Semen was diluted at 37°C with BioXcell extender (BioXcell, West Lebanon, NH) to a final concentration of 30×106 spermatozoa mL−1, and straws were kept at 4°C for 4h and then frozen in an automated system. After thawing, Percoll-separated spermatozoa were incubated at 37°C for 1h with 0, 20, and 100µM ZVAD-FMK. Sperm viability and membrane integrity were assessed by Trypan Blue/Giemsa and hypo-osmotic swelling test, respectively, as previously described (Longobardi et al. 2017 Theriogenology 88, 1-8). Sperm motility was examined by phase contrast microscopy at 40× magnification on a thermoregulated stage at 37°C. Apoptosis was evaluated by TUNEL technique, which assesses DNA fragmentation (Takeda et al. 201561, 185-190). The mitochondrial membrane potential was then assessed by flow cytometric analysis with the mitochondrial probe JC-1 (Garner and Thomas 1999Mol. Reprod. Dev. 53, 222-229). Data were analysed by ANOVA using least significant difference as post-hoc test. The treatment of bovine frozen-thawed sperm with 100µM ZVAD-FMK decreased the percentage of sperm exhibiting DNA fragmentation (17.8±1.1, 13.3±2.8, and 10.5±2.5 with 0, 20, and 100µM ZVAD, respectively; P&lt;0.05). Moreover, both concentrations of ZVAD-FMK increased the percentage of hypo-osmotic swelling test+ sperm, indicating improved membrane integrity compared with the control (60.5±3.5, 70.9±2.1, and 74.3±2.1 with 0, 20, and 100µM ZVAD-FMK, respectively; P&lt;0.01). However, no differences were found in sperm viability (82.3±0.5, 84.6±1.0, and 84.3±2.1 with 0, 20, and 100µM ZVAD-FMK, respectively) and motility (60.0±2.9, 62.5±3.4, and 67.5±2.1 with 0, 20, and 100µM ZVAD-FMK, respectively). Furthermore, no differences were observed among groups in the percentage of sperm exhibiting normal mitochondrial membrane potential (62.4±12.7, 57.9±12.8, and 50.8±8.8 with 0, 20, and 100µM ZVAD, respectively). In conclusion, caspase inhibition with 100µM ZVAD-FMK after thawing was effective in reducing sperm DNA fragmentation and increasing sperm membrane integrity, suggesting a beneficial effect on fertility. However, as the other fertility-related parameters did not improve, further investigations are required to draw definite conclusions.

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