Effect of donkey seminal plasma on sperm movement and sperm–polymorphonuclear neutrophils attachment in vitro

Seminal plasma (SP) is known to have immunosuppressive properties in several species. Equine SP has been reported to reduce or inhibit chemotaxis, phagocytosis and complement activity in vitro. The type and amount of the SP component that suppresses sperm–polymorphonuclear neutrophil (PMN) binding in vitro was determined, and the effect of such suppression on the fertility of mares inseminated in the presence of uterine inflammation, was analyzed. Sperm cells were suspended in either SP, semen extender or a mixture of both, and each was mixed with PMN-rich uterine secretions collected at 12 h after artificial insemination (AI). SP reduced binding between spermatozoa and PMNs significantly (P < 0.05). Fertile spermatozoa were suspended in SP or semen extender and used to inseminate mares 12 h after the induction of uterine inflammation. The pregnancy rate was normal (77%) when spermatozoa were suspended in SP, but was dramatically reduced to only 5% when spermatozoa were suspended in extender. The proteins from SP, blood plasma (BP) and a skim-milk-based semen extender (skim milk extender, SME) were precipitated by ammonium sulfate, resuspended in PBS and dialyzed. The effect of the precipitated proteins on sperm–PMN binding was compared with fresh, untreated SP. Both fresh SP, and isolated SP proteins reduced sperm–PMN binding (P < 0.001). Conversely, proteins isolated from either BP or SME did not reduce sperm–PMN binding. The different concentrations of SP proteins used showed a dose-dependent suppression of sperm–PMN binding. Concentrations of 1 mg/ml SP protein significantly reduced sperm–PMN binding and 6 mg/ml reduced the binding to a level similar to that observed with fresh whole SP (P < 0.001). Finally, SP protein digested with proteinase K resulted in the complete loss of SP suppressive activity confirming that the effective component is a proteinaceous substance.

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Seminal Plasma, Sperm Concentration, and Sperm-PMN Interaction in the Donkey: An In Vitro Model to Study Endometrial Inflammation at Post-Insemination

International Journal of Molecular Sciences ◽

10.3390/ijms21103478 ◽

2020 ◽

Vol 21 (10) ◽

pp. 3478 ◽

Cited By ~ 3

Author(s):

Jordi Miró ◽

Henar Marín ◽

Jaime Catalán ◽

Marion Papas ◽

Sabrina Gacem ◽

...

Keyword(s):

Seminal Plasma ◽

Artificial Insemination ◽

In Vitro Model ◽

Sperm Concentration ◽

Polymorphonuclear Neutrophils ◽

Low Fertility ◽

Fertility Rates ◽

Vitro Model ◽

Instrumental Role

In the donkey, artificial insemination (AI) with frozen-thawed semen is associated with low fertility rates, which could be partially augmented through adding seminal plasma (SP) and increasing sperm concentration. On the other hand, post-AI endometrial inflammation in the jenny is significantly higher than in the mare. While previous studies analyzed this response through recovering Polymorphonuclear Neutrophils (PMN) from uterine washings, successive lavages can detrimentally impact the endometrium, leading to fertility issues. For this reason, the first set of experiments in this work intended to set an in vitro model through harvesting PMN from the peripheral blood of jennies. Thereafter, how PMN, which require a triggering agent like formyl-methionyl-leucyl-phenylalanine (FMLP) to be activated, are affected by donkey semen was interrogated. Finally, we tested how four concentrations of spermatozoa (100 × 106, 200 × 106, 500 × 106 and 1000 × 106 spermatozoa/mL) affected their interaction with PMN. We observed that semen, which consists of sperm and SP, is able to activate PMN. Whereas there was a reduced percentage of spermatozoa phagocytosed by PMN, most remained attached on the PMN surface or into a surrounding halo. Spermatozoa not attached to PMN were viable, and most of those bound to PMN were also viable and showed high tail beating. Finally, only sperm concentrations higher than 500 × 106 spermatozoa/mL showed free sperm cells after 3 h of incubation, and percentages of spermatozoa not attached to PMN were higher at 3 h than at 1 h, exhibiting high motility. We can thus conclude that semen activates PMN in the donkey, and that the percentage of spermatozoa phagocytosed by PMN is low. Furthermore, because percentages of spermatozoa not attached to PMN were higher after 3 h than after 1 h of incubation, we suggest that PMN-sperm interaction plays an instrumental role in the reproductive strategy of the donkey.

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Effects of seminal plasma and semen application during in vitro fertilization treatment - systematic review and meta-ananlysis

10.26226/morressier.5912d9e7d462b802923868d8 ◽

2017 ◽

Author(s):

Marialuigia Spinelli

Keyword(s):

Systematic Review ◽

In Vitro Fertilization ◽

Seminal Plasma ◽

Vitro Fertilization

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Seminal Plasma: Relevant for Fertility?

International Journal of Molecular Sciences ◽

10.3390/ijms22094368 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4368

Author(s):

Heriberto Rodriguez-Martinez ◽

Emilio A. Martinez ◽

Juan J. Calvete ◽

Fernando J. Peña Vega ◽

Jordi Roca

Keyword(s):

Seminal Plasma ◽

Current Knowledge ◽

Inorganic Ions ◽

Vitro Fertilization ◽

Dna And Rna ◽

Model Animal ◽

Sexual Glands ◽

Species Specific

Seminal plasma (SP), the non-cellular component of semen, is a heterogeneous composite fluid built by secretions of the testis, the epididymis and the accessory sexual glands. Its composition, despite species-specific anatomical peculiarities, consistently contains inorganic ions, specific hormones, proteins and peptides, including cytokines and enzymes, cholesterol, DNA and RNA—the latter often protected within epididymis- or prostate-derived extracellular vesicles. It is beyond question that the SP participates in diverse aspects of sperm function pre-fertilization events. The SP also interacts with the various compartments of the tubular genital tract, triggering changes in gene function that prepares for an eventual successful pregnancy; thus, it ultimately modulates fertility. Despite these concepts, it is imperative to remember that SP-free spermatozoa (epididymal or washed ejaculated) are still fertile, so this review shall focus on the differences between the in vivo roles of the SP following semen deposition in the female and those regarding additions of SP on spermatozoa handled for artificial reproduction, including cryopreservation, from artificial insemination to in vitro fertilization. This review attempts, including our own results on model animal species, to critically summarize the current knowledge of the reproductive roles played by SP components, particularly in our own species, which is increasingly affected by infertility. The ultimate goal is to reconcile the delicate balance between the SP molecular concentration and their concerted effects after temporal exposure in vivo. We aim to appraise the functions of the SP components, their relevance as diagnostic biomarkers and their value as eventual additives to refine reproductive strategies, including biotechnologies, in livestock models and humans.

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Cannabidiol (CBD) Alters the Functionality of Neutrophils (PMN). Implications in the Refractory Epilepsy Treatment

Pharmaceuticals ◽

10.3390/ph14030220 ◽

2021 ◽

Vol 14 (3) ◽

pp. 220

Author(s):

Claudia Taborda Gómez ◽

Fabiana Lairion ◽

Marisa Repetto ◽

Miren Ettcheto ◽

Amalia Merelli ◽

...

Keyword(s):

Refractory Epilepsy ◽

Polymorphonuclear Neutrophils ◽

Dependent Manner ◽

Nuclear Condensation ◽

Concentration Dependent Manner ◽

Superoxide Anion Production ◽

Excretory Function ◽

Psychoactive Effects ◽

Stress Damage

Cannabidiol (CBD), a lipophilic cannabinoid compound without psychoactive effects, has emerged as adjuvant of anti-epileptic drugs (AEDs) in the treatment of refractory epilepsy (RE), decreasing the severity and/or frequency of seizures. CBD is considered a multitarget drug that could act throughout the canonical endocannabinoid receptors (CB1-CB2) or multiple non-canonical pathways. Despite the fact that the CBD mechanism in RE is still unknown, experiments carried out in our laboratory showed that CBD has an inhibitory role on P-glycoprotein excretory function, highly related to RE. Since CB2 is expressed mainly in the immune cells, we hypothesized that CBD treatment could alter the activity of polymorphonuclear neutrophils (PMNs) in a similar way that it does with microglia/macrophages and others circulating leukocytes. In vitro, CBD induced PMN cytoplasmatic vacuolization and proapoptotic nuclear condensation, associated with a significantly decreased viability in a concentration-dependent manner, while low CBD concentration decreased PMN viability in a time-dependent manner. At a functional level, CBD reduced the chemotaxis and oxygen consumption of PMNs related with superoxide anion production, while the singlet oxygen level was increased suggesting oxidative stress damage. These results are in line with the well-known CBD anti-inflammatory effect and support a potential immunosuppressor role on PMNs that could promote an eventual defenseless state during chronic treatment with CBD in RE.

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Specific Seminal Plasma Fractions Are Responsible for the Modulation of Sperm–PMN Binding in the Donkey

Animals ◽

10.3390/ani11051388 ◽

2021 ◽

Vol 11 (5) ◽

pp. 1388

Author(s):

Jordi Miró ◽

Jaime Catalán ◽

Henar Marín ◽

Iván Yánez-Ortiz ◽

Marc Yeste

Keyword(s):

Molecular Weight ◽

Sperm Motility ◽

Seminal Plasma ◽

Potential Effect ◽

Polymorphonuclear Neutrophils ◽

Low Fertility ◽

Plasma Fractions ◽

Complex Fluid ◽

First Time

While artificial insemination (AI) with frozen-thawed sperm results in low fertility rates in donkeys, the addition of seminal plasma, removed during cryopreservation, partially counteracts that reduction. Related to this, an apparent inflammatory reaction in jennies is induced following AI with frozen-thawed sperm, as a high amount of polymorphonuclear neutrophils (PMN) are observed within the donkey uterus six hours after AI. While PMN appear to select the sperm that ultimately reach the oviduct, two mechanisms, phagocytosis and NETosis, have been purported to be involved in that clearance. Remarkably, sperm interacts with PMN, but the presence of seminal plasma reduces that binding. As seminal plasma is a complex fluid made up of different molecules, including proteins, this study aimed to evaluate how different seminal plasma fractions, separated by molecular weight (<3, 3–10, 10–30, 30–50, 50–100, and >100 kDa), affect sperm–PMN binding. Sperm motility, viability, and sperm–PMN binding were evaluated after 0 h, 1 h, 2 h, 3 h, and 4 h of co-incubation at 38 °C. Two seminal plasma fractions, including 30–50 kDa or 50–100 kDa proteins, showed the highest sperm motility and viability. As viability of sperm not bound to PMN after 3 h of incubation was the highest in the presence of 30–50 and 50–100 kDa proteins, we suggest that both fractions are involved in the control of the jenny’s post-breeding inflammatory response. In conclusion, this study has shown for the first time that specific fractions rather than the entire seminal plasma modulate sperm–PMN binding within the donkey uterus. As several proteins suggested to be involved in the control of post-AI endometritis have a molecular weight between 30 and 100 kDa, further studies aimed at determining the identity of these molecules and evaluating their potential effect in vivo are much warranted.

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In Vitro Anti-Inflammatory and Immunomodulatory Activities of an Extract from the Roots of Bupleurum rotundifolium

Medicines ◽

10.3390/medicines6040101 ◽

2019 ◽

Vol 6 (4) ◽

pp. 101 ◽

Cited By ~ 2

Author(s):

Cholet ◽

Decombat ◽

Vareille-Delarbre ◽

Gainche ◽

Berry ◽

...

Keyword(s):

Mononuclear Cells ◽

Polymorphonuclear Neutrophils ◽

Ros Production ◽

Peripheral Blood Mononuclear ◽

Aerial Parts ◽

Bupleurum Scorzonerifolium Willd ◽

Anti Inflammatory ◽

Inflammatory Profile ◽

Bupleurum Scorzonerifolium

Background: Some Bupleurum species, such as the Bupleurum chinense DC. or the Bupleurum scorzonerifolium Willd have been extensively studied (especially their roots) for the treatment of inflammation. In contrast, only compounds extracted from the aerial parts of Bupleurum rotundifolium have been studied and showed anti-inflammatory or antiproliferative activities. This study was conducted to investigate the antioxidant, anti-inflammatory, and immunomodulatory effects of Bupleurum rotundifolium roots. Methods: To tackle the various aspects of inflammation, we studied in vitro a methanolic extract from the roots of Bupleurum rotundifolium on peripheral blood mononuclear cells (PBMCs), polymorphonuclear neutrophils (PMNs), and the monocytic cells THP-1. Its antioxidant capacities and iron-chelating activity were assessed. The extract was tested on THP-1 differentiation, reactive oxygen species (ROS) production by leukocytes, neutrophils chemotaxis, cytokines, PGE2 production, and NF-κB activation in PBMCs. Results: The extract showed a decreased ROS production in stimulated cells. It increased PBMC chemokine secretion and up-regulated the differentiation of THP-1 monocytes into macrophage-like cells, indicating a potential interest of the extract in the resolution of acute inflammation. In addition, the analysis of cytokine production suggests that Bupleurum rotundifolium has immunomodulatory properties. Conclusions: Cytokines secretion, especially IL-1β and IL-12p70, provided us with a set of indicators suggesting that the extract might be able to drive the polarization of macrophages and lymphocytes toward a Th2 anti-inflammatory profile in excessive inflammation.

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