The Presence of Seminal Plasma during Liquid Storage of Pig Spermatozoa at 17 °C Modulates Their Ability to Elicit In Vitro Capacitation and Trigger Acrosomal Exocytosis

Although seminal plasma is essential to maintain sperm integrity and function, it is diluted/removed prior to liquid storage and cryopreservation in most mammalian species. This study sought to evaluate, using the pig as a model, whether storing semen in the presence of seminal plasma affects the sperm ability to elicit in vitro capacitation and acrosomal exocytosis. Upon collection, seminal plasma was separated from sperm samples, which were diluted in a commercial extender, added with seminal plasma (15% or 30%), and stored at 17 °C for 48 or 72 h. Sperm cells were subsequently exposed to capacitating medium for 4 h, and then added with progesterone to induce acrosomal exocytosis. Sperm motility, acrosome integrity, membrane lipid disorder, intracellular Ca2+ levels, mitochondrial activity, and tyrosine phosphorylation levels of glycogen synthase kinase-3 (GSK3)α/β were determined after 0, 2, and 4 h of incubation, and after 5, 30, and 60 min of progesterone addition. Results showed that storing sperm at 17 °C with 15% or 30% seminal plasma led to reduced percentages of viable spermatozoa exhibiting an exocytosed acrosome, mitochondrial membrane potential, intracellular Ca2+ levels stained by Fluo3, and tyrosine phosphorylation levels of GSK3α/β after in vitro capacitation and progesterone-induced acrosomal exocytosis. Therefore, the direct contact between spermatozoa and seminal plasma during liquid storage at 17 °C modulated their ability to elicit in vitro capacitation and undergo acrosomal exocytosis, via signal transduction pathways involving Ca2+ and Tyr phosphorylation of GSK3α/β. Further research is required to address whether such a modulating effect has any impact upon sperm fertilizing ability.

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HVCN1 Channels Are Relevant for the Maintenance of Sperm Motility During In Vitro Capacitation of Pig Spermatozoa

International Journal of Molecular Sciences ◽

10.3390/ijms21093255 ◽

2020 ◽

Vol 21 (9) ◽

pp. 3255

Author(s):

Marc Yeste ◽

Marc Llavanera ◽

Yentel Mateo-Otero ◽

Jaime Catalán ◽

Sergi Bonet ◽

...

Keyword(s):

Membrane Potential ◽

Sperm Motility ◽

Mitochondrial Membrane ◽

Membrane Lipid ◽

Sperm Head ◽

Sperm Viability ◽

Lipid Disorder ◽

Acrosomal Exocytosis ◽

Control Samples

The objective of the present study was to determine the physiological role of voltage-gated hydrogen channels 1 (HVCN1 channels) during in vitro capacitation of pig spermatozoa. Sperm samples from 20 boars were incubated in capacitating medium for 300 minutes (min) in the presence of 2-guanidino benzimidazole (2-GBI), a specific HVCN1-channel blocker, added either at 0 min or after 240 min of incubation. Control samples were incubated in capacitating medium without the inhibitor. In all samples, acrosomal exocytosis was triggered with progesterone after 240 min of incubation. Sperm viability, sperm motility and kinematics, acrosomal exocytosis, membrane lipid disorder, intracellular calcium levels and mitochondrial membrane potential were evaluated after 0, 60, 120, 180, 240, 250, 270 and 300 min of incubation. While HVCN1-blockage resulted in altered sperm viability, sperm motility and kinematics and reduced mitochondrial membrane potential as compared to control samples, at any blocker concentration and incubation time, it had a non-significant effect on intracellular Ca2+ levels determined through Fluo3-staining. The effects on acrosomal exocytosis were only significant in blocked samples at 0 min, and were associated with increased membrane lipid disorder and Ca2+ levels of the sperm head determined through Rhod5-staining. In conclusion, HVCN1 channels play a crucial role in the modulation of sperm motility and kinematics, and in Ca2+ entrance to the sperm head.

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Blocking NHE Channels Reduces the Ability of In Vitro Capacitated Mammalian Sperm to Respond to Progesterone Stimulus

International Journal of Molecular Sciences ◽

10.3390/ijms222312646 ◽

2021 ◽

Vol 22 (23) ◽

pp. 12646

Author(s):

Marc Yeste ◽

Sandra Recuero ◽

Carolina Maside ◽

Albert Salas-Huetos ◽

Sergi Bonet ◽

...

Keyword(s):

Plasma Membrane ◽

Mammalian Species ◽

Physiological Role ◽

Membrane Lipid ◽

Equatorial Region ◽

Lipid Disorder ◽

Head Displacement ◽

Mammalian Sperm ◽

Few Data

Few data exist about the presence and physiological role of Na+/H+ exchangers (NHEs) in the plasma membrane of mammalian sperm. In addition, the involvement of these channels in the ability of sperm to undergo capacitation and acrosomal reaction has not been investigated in any mammalian species. In the present study, we addressed whether these channels are implicated in these two sperm events using the pig as a model. We also confirmed the presence of NHE1 channels in the plasma membrane of ejaculated sperm by immunofluorescence and immunoblotting. The function of NHE channels during in vitro capacitation was analyzed by incubating sperm samples in capacitating medium for 300 min in the absence or presence of a specific blocker (DMA; 5-(N,N-dimethyl)-amiloride) at different concentrations (1, 5, and 10 µM); acrosome exocytosis was triggered by adding progesterone after 240 min of incubation. Sperm motility and kinematics, integrity of plasma and acrosome membranes, membrane lipid disorder, intracellular calcium and reactive oxygen species (ROS) levels, and mitochondrial membrane potential (MMP) were evaluated after 0, 60, 120, 180, 240, 250, 270, and 300 min of incubation. NHE1 localized in the connecting and terminal pieces of the flagellum and in the equatorial region of the sperm head and was found to have a molecular weight of 75 kDa. During the first 240 min of incubation, i.e., before the addition of progesterone, blocked and control samples did not differ significantly in any of the parameters analyzed. However, from 250 min of incubation, samples treated with DMA showed significant alterations in total motility and the amplitude of lateral head displacement (ALH), acrosomal integrity, membrane lipid disorder, and MMP. In conclusion, while NHE channels are not involved in the sperm ability to undergo capacitation, they could be essential for triggering acrosome exocytosis and hypermotility after progesterone stimulus.

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92 EVALUATION OF BOAR SPERM FUNCTIONALITY AFTER A CUSHIONED CENTRIFUGATION TECHNIQUE

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab92 ◽

2006 ◽

Vol 18 (2) ◽

pp. 154

Author(s):

J. Gadea ◽

S. Martínez-Miró ◽

G. Decuadro-Hansen ◽

C. Matás

Keyword(s):

Seminal Plasma ◽

Acrosome Reaction ◽

Sperm Viability ◽

Lipid Packing ◽

Lipid Disorder ◽

Merocyanine 540 ◽

Centrifuge Tube ◽

Before And After ◽

Centrifugation Technique

Separation of sperm from seminal plasma is required in most semen freezing procedures. Semen is typically subjected to centrifugation to concentrate sperm into a pellet and allow removal of the seminal plasma prior to dilution in freezing extender. Centrifugation is a relatively effective method to recover sperm, however, the process also causes considerable sperm damage. The use of a dense, inert, and isotonic solution as a cushion in the bottom of the centrifuge tube allows a greater centrifugation speed to be applied and results in greater sperm recovery. The aim of the present work was to evaluate the effects of this cushioned centrifugation technique on in vitro sperm viability and functionality. Sperm-rich fractions from 16 fertile boars were diluted and cooled to 15�C; then subsamples were centrifuged by one of two different techniques. A standard method (SM), 800 g for 10 min in 50-mL tubes (Westendorf et al. 1975 Dtsch. Tier�rztl. Wschr. 82, 261-267) and a cushioned method (CM), 1000 g for 20 min using 45 mL of diluted semen on 5 mL of an isotonic iodixanol solution (60% w/v gradient) were performed. Sperm samples were stained with merocyanine 540 (M540) and Yo-Pro 1 (Harrison et al. 1996 Mol. Rep. Dev. 45, 378-391) to detect changes in lipid packing disorder of the plasma membrane. Another set of sperm samples was incubated in the presence of (0.7 �M) 22,72-dichlorodihydrofluorescein diacetate (Gadea et al. 2005 J. Androl. 26, 396-404) to estimate production of reactive oxygen species (ROS). A final set of sperm samples was stained with peanut aggultinin-fluorscein isothiocyanate (PNA-FITC) and propidium iodide to evaluate the acrosome reaction. All of these parameters were evaluated by flow cytometry before and after centrifugation. ANOVA analysis revealed that centrifugation altered lipid packing disorder and viability. Raw semen (RS) had a larger number of viable low lipid disorder sperm than centrifuged semen (RS = 86.9a vs. SM = 81.64b vs. CM = 80.6b, P < 0.01) and a decreased number of dead sperm cells (RS = 9.5a vs. SM = 15.0b vs. CM = 16.3b, P < 0.01). However, the cushioned and standard centrifugation methods yielded similar results for all the parameters measured. No significant differences were found for generation of ROS or in the number of sperm exhibiting the acrosome reaction. In conclusion, compared to the standard centrifugation method, this simple cushioned modification is a more efficient means of processing boar semen for freezing because significantly less sample losses are detected; also, it provides similar levels of sperm viability and functionality, and consequently a higher number of doses per ejaculation can be produced.

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Impact of Cryopreservation on Motile Subpopulations and Tyrosine-Phosphorylated Regions of Ram Spermatozoa during Capacitating Conditions

Biology ◽

10.3390/biology10111213 ◽

2021 ◽

Vol 10 (11) ◽

pp. 1213

Author(s):

Patricia Peris-Frau ◽

Irene Sánchez-Ajofrín ◽

Alicia Martín Maestro ◽

Carolina Maside ◽

Daniela Alejandra Medina-Chávez ◽

...

Keyword(s):

Tyrosine Phosphorylation ◽

Mitochondrial Activity ◽

Strongly Correlated ◽

Motile Sperm ◽

Imaging Flow Cytometry ◽

Heterogeneous Nature ◽

Key Events ◽

Casa System ◽

The Relationship

The heterogeneous nature of ejaculates highlights the relevance of studying the behavior of different sperm subpopulations. Changes in sperm motility and the increase in tyrosine phosphorylation are key events that usually occur during capacitation and can be modified by the cryopreservation process. However, the relationship between both events remains poorly defined throughout capacitation in the different sperm subpopulations. Fresh and frozen-thawed spermatozoa were incubated in capacitating (CAP) and non-capacitating (NC) media up to 240 min. Sperm kinematics, tyrosine phosphorylation and mitochondrial activity were measured by the CASA system and imaging flow cytometry. Four motile sperm subpopulations (SP) were identified in fresh and frozen-thawed ram semen after the cluster analysis. Incubation under CAP conditions over time led to greater changes in the percentage of spermatozoa included in each subpopulation compared to NC conditions, being different between fresh and frozen-thawed spermatozoa. The SP1, characterized by slow spermatozoa, progressively increased after 15 min in frozen-thawed samples incubated in both media but not in fresh ones. The SP4, characterized by fast and non-linear spermatozoa, showed a marked increase during CAP, but not under NC conditions, occurring more rapidly in frozen-thawed spermatozoa. This subpopulation (SP4) was also the only one positively and strongly correlated with mitochondrial activity and all phosphorylated sperm regions during capacitation, either in fresh or frozen-thawed samples. Our results indicated that in vitro capacitation induced significant changes in the distribution of motile sperm subpopulations, being affected by cryopreservation. Notwithstanding, the subpopulation which probably represents hyperactivated-like spermatozoa (SP4) also increased in frozen-thawed samples, occurring faster and simultaneously to the increment of mitochondrial activity and tyrosine phosphorylation of different sperm regions.

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TLR7/8 Signalling Affects X-Sperm Motility via the GSK3 α/β-Hexokinase Pathway for the Efficient Production of Sexed Dairy Goat Embryos

10.21203/rs.3.rs-227816/v1 ◽

2021 ◽

Author(s):

Fa Ren ◽

Huaming Xi ◽

Yijie Ren ◽

Yu Li ◽

Fei Wen ◽

...

Keyword(s):

Flow Cytometry ◽

Sperm Motility ◽

Cross Sections ◽

Glycogen Synthase ◽

Lower Layer ◽

Economic Benefits ◽

Dairy Goat ◽

Mitochondrial Activity ◽

Efficient Production

Abstract BackgroundGoat milk is most similar to human milk because of its abundant nutrients and ease of digestion. To derive more economic benefits, farmers need to obtain offspring with more does; however, the buck to doe sex ratio of offspring is approximately 50%. At present, artificial insemination after separation of X/Y sperm using flow cytometry is the primary means to control the sex of livestock offspring. However, flow cytometry has not been successful in separating X/Y sperm for sexing control in dairy goats.ResultsIn this study, a novel, simple goat sperm-sexing technology that activates the toll-like receptor 7/8 (TLR7/8), inhibiting X-sperm motility, was investigated. Results showed that TLR7/8 coding goat X-chromosome was expressed in approximately 50% of round spermatids in testis' cross-sections and sperm in the epididymis' cross-sections and ejaculate. Importantly, TLR7/8 was located at the tail of X-sperm. Upon TLR7/8 activation, phosphorylated forms of glycogen synthase kinase α/β (GSK3 α/β) and nuclear factor kappa-B (NFκB) were detected in the X-sperm, reducing mitochondrial activity, ATP levels, and sperm motility. High-motility Y-sperm segregated to the upper layer and low-motility X-sperm to the lower layer. Following in vitro fertilisation using the lower layer TLR7/8-activated sperm, 80.52 ± 6.75% of the embryos were XX females. TLR7/8-activated sperm was used for in vivo embryo production via the superovulatory response, and 88.89% of the embryos were XX females.ConclusionsOur study reveals a novel TLR7/8 signalling mechanism that affects X-sperm motility via the GSK3 α/β-hexokinase pathway, facilitating the efficient production of sexed dairy goat embryos.

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326 COMPARING CHANGES IN MEMBRANE LIPID ORDER IN EPIDIDYMAL AND EJACULATED BOAR SPERMATOZOA

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab326 ◽

2007 ◽

Vol 19 (1) ◽

pp. 277

Author(s):

C. Matas ◽

F. Garcia-Vazquez, ◽

M. Sansegundo ◽

S. Ruiz ◽

J. Gadea

Keyword(s):

Plasma Membrane ◽

Seminal Plasma ◽

Lipid Membrane ◽

Calcium Influx ◽

Membrane Lipid ◽

Treatment Groups ◽

Lipid Disorder ◽

Lipid Order ◽

Merocyanine 540 ◽

Boar Spermatozoa

The diffusion of lipids in the plasma membrane of ejaculated spermatozoa is influenced by seminal plasma proteins and the composition of the suspending medium (Wolfe et al. 2001 Mol. Reprod. Dev. 59, 306–313). Merocyanine 540 (M540) is a hydrophobic dye that has been shown to stain cell membranes more intensely if their lipid components are in a higher state of disorder, as is the case of capacitated spermatozoa. It is believed that the membrane fluidity changes detected by M540 precede the calcium influx, making M540 a method for evaluating the early events of capacitation. The aim of this study was to determine if there are differences in the dynamics of lipid disorder in the plasma membrane of ejaculated and epididymal boar spermatozoa under different conditions of capacitation. The sperm capacitation treatments were: washed in Delbucco's PBS supplemented with 0.1 % BSA (PBS-BSA), washed on a Percoll gradient (PG), and unwashed (UW: Control). During measurement, the samples were kept at 38�C and 5 % CO2 to maintain constant incubation conditions. Membrane lipid order and sperm viability were determined by flow cytometry with M540 (2.7 �M) and Yo-Pro-1 (25 nM), respectively. Samples were analyzed on a Coulter Epics XL flow cytometer (Beckman Coulter Co., Inc., Fullerton, CA, USA). A total of 10 000 gated events were collected per sample, with sample running rates of approximately 600 events/s. Data were analyzed by analysis of variance (ANOVA). For the epidydimal vs. ejaculated results, the percentage of low lipid disorder spermatozoa was higher in the epididymal (19.23%) than in the ejaculated (5.84%) groups, and the proportion of high disorder (42.85%) and dead cells (48.59%) was higher in the ejaculated group. In relation to sperm treatment (UW, PBS-BSA, and PG), the percentage of high disorder was similar in all of the treatment groups (UW: 44.62 %; PBS-BSA: 43.08%; PG: 43.41%). Finally, the percentage of low disorder was lower in the PBS-BSA and PERCOLL (10.68% and 12.83%, respectively) groups, and the highest was obtained for the UW group (14.09%). In conclusion, the staining with M540 revealed that the lipid disorder was affected by the source of the sperm and the sperm treatment. A significant increase in membrane lipid low disorder and decrease in high disorder and dead cells were detected when epididymal sperm were compared with ejaculated sperm, so the seminal plasma and the sperm treatment to eliminate disorder have an important effect in the lipid membrane order. Supported by MEC (AGL2006-03495/GAN) and Fundaci�n S�neca (03018/PI/05).

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329 COMPARING CHANGES IN MOTION PARAMETERS IN EPIDIDYMAL AND EJACULATED BOAR SPERMATOZOA UNDER THREE DIFFERENT TREATMENTS

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab329 ◽

2007 ◽

Vol 19 (1) ◽

pp. 280

Author(s):

M. Sansegundo ◽

J. C. Gardon ◽

F. Garcia-Vazquez ◽

J. Gadea ◽

C. Matas

Keyword(s):

Seminal Plasma ◽

Objective Assessment ◽

Mammalian Species ◽

Computer Assisted ◽

Sperm Analysis ◽

Motion Parameters ◽

Computer Assisted Sperm Analysis ◽

Boar Spermatozoa ◽

Curvilinear Trajectory

The motion ability of mammalian spermatozoa is acquired during their epididymal transit but observed only upon dilution with seminal plasma (SP) at the time of ejaculation (Yanahimachi 1994 in The Physiology of Reproduction, New York: Raven Press). The bicarbonate present in seminal plasma activates multiple sperm functions, some of which are essential for the initiation of motility. Sperm hyperactivity has been observed in vitro in various mammalian species, especially if capacitation of spermatozoa was induced with Ca2+ and bicarbonate media, such as TALP (Harrison et al. 1996 Mol. Reprod. Dev. 45, 378–391). Computer-assisted sperm analysis (CASA) is a tool for the objective assessment of sperm motility. The aim of this study was to determine if there are differences in motility parameters of ejaculated (EJ) and epididymal (EP) boar spermatozoa under different treatments. Ejaculated and epididymal sperm cells from 10 different boars in each group were used. The sperm treatments were: washed in Dulbecco's PBS supplemented with 0.1&percnt; BSA (PBS-BSA), washed on a Percoll gradient (PG), and unwashed (UW: Control); the sperm samples were incubated in TALP medium at 38.5°C and 5&percnt; CO2 during the analysis. Motion parameters were determined using a computer-assisted sperm analysis (CASA) system. A 7-µL drop of the sample was placed on a warmed (37°C) slide. At least 4 fields per sample were evaluated, with a minimum of 100 spermatozoa counted per sub-sample. The CASA-derived motility characteristics studied were motility (MOT, &percnt;), progressive motility (PM, &percnt;), curvilinear velocity (VCL, µm s−1), straight-line velocity (VSL, µm s−1), average path velocity (VAP, µm s−1), linearity of the curvilinear trajectory (LIN, ratio of VSL/VCL, &percnt;), straightness (STR, ratio of VSL/VAP, &percnt;), amplitude of lateral head displacement (ALH, µm), wobble of the curvilinear trajectory (WOB, ratio of VAP/VCL, &percnt;), and beat cross-frequency (BCF, Hz). Data were analyzed by ANOVA. If we evaluated all of the data together (EJ vs. EP), EP sperm after treatment showed a higher motility (PM: 38.20&percnt;; MOT: 74.23&percnt;) than EJ sperm (PM: 29.27&percnt;; MOT: 63.24&percnt;), and all of the motion parameters related to velocities and ALH were higher in EP (VCL: 86.02; VSL: 41; VAP: 57.94; ALH: 3.21) than in EJ (VCL: 69.70; VSL: 34.67; VAP: 48.16; ALH: 2.54). No differences were found for LIN, STR, WOB, and BCF. The treatments significantly affected the VCL and ALH, with lower values for the PG treatment. When VCL was lower and the VSL and VAP were similar, consequently the LIN and WOB were significantly higher for the PG group. STR also was higher for the PG group. In conclusion, when both groups of sperm were incubated in TALP medium, the EJ sperm showed a decrease in the majority of motion parameters when compared with EP sperm. This work was supported by MEC (AGL2006-03495/GAN) and Fundación Séneca (03018/PI/05).

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Effects of the environmental endocrine disruptors di-2-ethylhexyl phthalate and mono-2-ethylhexyl phthalate on human sperm function in vitro

Reproduction Fertility and Development ◽

10.1071/rd19164 ◽

2020 ◽

Vol 32 (6) ◽

pp. 629

Author(s):

Xinyi Sun ◽

Wenqiong Chen ◽

Shiqi Weng ◽

Tingting Pan ◽

Xiaonian Hu ◽

...

Keyword(s):

Tyrosine Phosphorylation ◽

Membrane Integrity ◽

Human Sperm ◽

Male Reproduction ◽

Human Spermatozoa ◽

Mitochondrial Activity ◽

Sperm Function ◽

Endocrine Disrupting Chemical ◽

Ethylhexyl Phthalate

Di-2-ethylhexyl phthalate (DEHP), a plastic-derived, endocrine-disrupting chemical, has been shown to exhibit male reproductive toxicity. However, its effects on human mature spermatozoa are largely unknown. In this study we investigated the invitro effects of DEHP and mono-2-ethylhexyl phthalate (MEHP; the main metabolite of DEHP) on sperm function and the mechanisms involved. Human spermatozoa were exposed to phthalates invitro at the doses that cover the concentrations detected in human semen: 20nM–8 μM DEHP, 1nM–20 μM MEHP or a mixture of 20nM–8 μM DEHP and 1nM–20 μM MEHP. DEHP and MEHP, alone or in combination, had no effect on the viability, membrane integrity, motility, homeostasis of reactive oxygen species or mitochondrial activity of human spermatozoa. Interestingly, 1nM–20 μM MEHP and combinations of 20nM–8 μM DEHP and 1nM–20 μM MEHP enhanced penetration ability, hyperactivation and the spontaneous acrosome reaction of human spermatozoa, and increased intracellular free Ca2+ concentrations ([Ca2+]i) and tyrosine phosphorylation, two key signalling pathways that regulate sperm function. The findings of this study suggest that invitro exposure to MEHP metabolised from DEHP affects human sperm function by inducing increases in sperm [Ca2+]i and tyrosine phosphorylation, which adds to our understanding of the effects of DEHP on male reproduction.

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Parkinson Disease Protein 7 (PARK7) Is Related to the Ability of Mammalian Sperm to Undergo In Vitro Capacitation

International Journal of Molecular Sciences ◽

10.3390/ijms221910804 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10804

Author(s):

Sandra Recuero ◽

Ariadna Delgado-Bermúdez ◽

Yentel Mateo-Otero ◽

Estela Garcia-Bonavila ◽

Marc Llavanera ◽

...

Keyword(s):

Oxidative Stress ◽

Parkinson Disease ◽

Intracellular Calcium ◽

Sperm Motility ◽

Antioxidant Properties ◽

Lipid Disorder ◽

Mammalian Sperm ◽

Acrosomal Exocytosis ◽

Disease Protein

Parkinson disease protein 7 (PARK7) is a multifunctional protein known to be involved in the regulation of sperm motility, mitochondrial function, and oxidative stress response in mammalian sperm. While ROS generation is needed to activate the downstream signaling pathways required for sperm to undergo capacitation, oxidative stress has detrimental effects for sperm cells and a precise balance between ROS levels and antioxidant activity is needed. Considering the putative antioxidant role of PARK7, the present work sought to determine whether this protein is related to the sperm ability to withstand in vitro capacitation. To this end, and using the pig as a model, semen samples were incubated in capacitation medium for 300 min; the acrosomal exocytosis was triggered by the addition of progesterone after 240 min of incubation. At each relevant time point (0, 120, 240, 250, and 300 min), sperm motility, acrosome and plasma membrane integrity, membrane lipid disorder, mitochondrial membrane potential, intracellular calcium and ROS were evaluated. In addition, localization and protein levels of PARK7 were also assessed through immunofluorescence and immunoblotting. Based on the relative content of PARK7, two groups of samples were set. As early as 120 min of incubation, sperm samples with larger PARK7 content showed higher percentages of viable and acrosome-intact sperm, lipid disorder and superoxide levels, and lower intracellular calcium levels when compared to sperm samples with lower PARK7. These data suggest that PARK7 could play a role in preventing sperm from undergoing premature capacitation, maintaining sperm viability and providing a better ability to keep ROS homeostasis, which is needed to elicit sperm capacitation. Further studies are required to elucidate the antioxidant properties of PARK7 during in vitro capacitation and acrosomal exocytosis of mammalian sperm, and the relationship between PARK7 and sperm motility.

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The Re-Addition of Seminal Plasma after Thawing Does Not Improve Buck Sperm Quality Parameters

Animals ◽

10.3390/ani11123452 ◽

2021 ◽

Vol 11 (12) ◽

pp. 3452

Author(s):

Uchechi Linda Ohaneje ◽

Uchebuchi Ike Osuagwuh ◽

Manuel Alvarez-Rodríguez ◽

Iván Yánez-Ortiz ◽

Abigail Tabarez ◽

...

Keyword(s):

Seminal Plasma ◽

Sperm Quality ◽

Membrane Integrity ◽

Egg Yolk ◽

Quality Parameters ◽

Mitochondrial Activity ◽

Dna Integrity ◽

Vitro Fertilization

In order to achieve a higher post-thaw buck sperm quality, an approach in the thawing protocol of cryopreserved sperm doses under in vitro capacitation conditions mimicking the in vivo female environment was studied. Therefore, functional and kinetic characteristics of buck thawed sperm from males of different ages, the season of collection, and melatonin implanted males in the non-breeding season were assessed after 3 h of incubation in an in vitro fertilization (IVF) media with 20% of buck seminal plasma (SP). Previously, fresh ejaculates were collected via artificial vagina from eight males of the Cabra Blanca de Rasquera breed during two consecutive years in breeding and non-breeding periods. Prior to semen collection in non-breeding seasons, males were split into two groups: one group was implanted with melatonin, while the other was not. In each group, semen samples were pooled, centrifuged, and diluted in an extender containing 15% powdered egg yolk and 5% glycerol before freezing. After thawing, sperm were washed and incubated in three different media: (a) control media (modified phosphate-buffered saline (PBS), (b) IVF commercial media, and (c) IVF media + 20% SP. Sperm motility was evaluated by CASA, while plasma and acrosome membrane integrity, mitochondria activity, and DNA fragmentation were analysed by flow cytometer at 0 h and after 3 h incubation. A significant reduction in motility, mitochondrial activity, plasma, and acrosome membrane integrity were observed after incubation in the presence of SP, although similar to that observed in IVF media alone. DNA integrity was not affected under in vitro capacitation conditions, regardless of SP addition. In conclusion, the addition of SP failed to improve post-thaw buck sperm quality under in vitro conditions irrespective of male age, the season of collection, and melatonin implant.

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