Aldose reductase B1 in pig seminal plasma: identification, localization in reproductive tissues, and relationship with quality and sperm preservation

This manuscript version is made available under the CC-BY 4.0 license http://creativecommons.org/licenses/by/4.0/. This document is the Published version of a Published Work that appeared in final form in Frontiers in Cell and Developmental Biology. To access the final edited and published work see https://doi.org/10.3389/fcell.2021.683199Aldose reductase B1 (AKR1B1), a NADPH-dependent enzyme that belongs to the aldo-keto reductase protein superfamily, has been reported to be involved in both male and female reproductive physiology. The objectives of this study were: (1) to evaluate the concentration of SP-AKR1B1 in pig ejaculate fractions; (2) to describe the immunohistochemical localization of AKR1B1 alongside the boar genital tract; (3) to evaluate the relationship between SP-AKR1B1 and sperm quality/functionality parameters. Ejaculates from seven boars (one ejaculate per boar) were collected in separate portions [the first 10 mL of the sperm rich fraction (SRF-P1), the rest of the SRF (SRF-P2), and the post-SRF (PSRF)], and the concentration of SP-AKR1B1 was assessed using an enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry and immunoblotting targeting was conducted in the reproductive tissues of these boars. Additionally, the entire ejaculates of 14 boars (one ejaculate per boar) were collected and split into three separate aliquots for: (i) SP-AKR1B1 quantification; (ii) assessment of sperm concentration and morphology; and (iii) evaluation of sperm quality and functionality parameters upon ejaculate collection (0 h) and after 72 h of liquid storage at 17°C. Concentration of AKR1B1 in the SP of SRF-P1 (458.2 ± 116.33 ng/mL) was lower (P < 0.05) than that of SRF-P2 (1105.0 ± 229.80 ng/mL) and PSRF (1342.4 ± 260.18 ng/mL). Monomeric and dimeric AKR1B1 forms were expressed alongside the reproductive tissues, except in the bulbourethral glands. No relationship between SP-AKR1B1 and sperm quality/functionality parameters was observed either at 0 h or after 72 h of storage at 17°C. In conclusion, AKR1B1 is expressed in the reproductive organs of boars (except bulbourethral glands) and a higher concentration is found in the PSRF suggesting that seminal vesicles would be the main secretory source. However, this enzyme does not appear to be related to sperm quality/functionality or to the sperm ability to withstand liquid storage at 17°C

Exploring seminal plasma GSTM3 as a quality and in vivo fertility biomarker in pigs-relationship with sperm morphology

© 2020 by the authors.This manuscript version is made available under the CC-BY 4.0 license http://creativecommons.org/licenses/by/4.0/ This document is the Published version of a Published Work that appeared in final form in Antioxidants. To access the final edited and published work see https://doi.org/10.3390/antiox9080741Glutathione S-transferases Mu 3 (GSTM3) is an essential antioxidant enzyme whose presence in sperm has recently been related to sperm cryotolerance, quality and fertility. However, its role in seminal plasma (SP) as a predictor of the same sperm parameters has never been investigated. Herein, cell biology and proteomic approaches were performed to explore the presence, origin and role of SP-GSTM3 as a sperm quality and in vivo fertility biomarker. GSTM3 in SP was quantified using a commercial Enzyme-Linked Immunosorbent Assay (ELISA) kit specific for Sus scrofa, whereas the presence of GSTM3 in testis, epididymis and accessory sex glands was assessed through immunoblotting analysis. Sperm quality and functionality parameters were evaluated in semen samples at 0 and 72 h of liquid-storage, whereas fertility parameters were recorded over a 12-months as farrowing rate and litter size. The presence and concentration of GSTM3 in SP was established for the first time in mammalian species, predominantly synthesized in the epididymis. The present study also evidenced a relationship between SP-GSTM3 and sperm morphology and suggested it is involved in epididymal maturation rather than in ejaculated sperm physiology. Finally, the data reported herein ruled out the role of this antioxidant enzyme as a quality and in vivo fertility biomarker of pig sperm

Seminal extracellular vesicles alter porcine in vitro fertilization outcome by modulating sperm metabolism

© This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/. This document is the Published version of a Published Work that appeared in final form in Theriogenology. To access the final edited and published work see https://doi.org/10.1016/j.theriogenology.2024.02.024Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEVs) that modulate several reproductive-related processes. This study investigated the effect of two sEV subsets, small (S-sEVs) and large (L-sEVs), on porcine in vitro fertilization (IVF). The sEVs were isolated from nine SP pools (five ejaculates/pool) using a size-exclusion chromatography-based procedure and characterized for quantity (total protein), morphology (cryogenic electron microscopy), size distribution (dynamic light scattering), purity and EV-protein markers (flow cytometry; albumin, CD81, HSP90β). The characterization confirmed the existence of two subsets of high purity (low albumin content) sEVs that differed in size (S- and L-sEVs). In vitro fertilization was performed with in vitro matured oocytes and frozen-thawed spermatozoa and the IVF medium was supplemented during gamete coincubation (1 h at 38.5 °C, 5 % CO2 in a humidified atmosphere) with three different concentrations of each sEV subset: 0 (control, without sEVs), 0.1, and 0.2 mg/mL. The first experiment showed that sEVs, regardless of subset and concentration, decreased penetration rates and total IVF efficiency (P < 0.0001). In a subsequent experiment, it was shown that sEVs, regardless of subset and concentration, impaired the ability of spermatozoa to bind to the zona pellucida of oocytes (P < 0.0001). The following experiment showed that sEVs, regardless of the subset, bound to frozen-thawed sperm but not to in vitro matured oocytes, indicating that sEVs would affect sperm functionality but not oocyte functionality. The lack of effect on oocytes was confirmed by incubating sEVs with oocytes prior to IVF, achieving sperm-zona pellucida binding results similar to those of control. In the last experiment, conducted under IVF conditions, sperm functionality was analyzed in terms of tyrosine phosphorylation, acrosome integrity and metabolism. The sEVs, regardless of the subset, did not affect sperm tyrosine phosphorylation or acrosome integrity, but did influence sperm metabolism by decreasing sperm ATP production under capacitating conditions. In conclusion, this study demonstrated that the presence of sEVs on IVF medium impairs IVF outcomes, most likely by altering sperm metabolism

Red-Light Irradiation of Horse Spermatozoa Increases Mitochondrial Activity and Motility through Changes in the Motile Sperm Subpopulation Structure

Previous studies in other mammalian species have shown that stimulation of semen with red-light increases sperm motility, mitochondrial activity, and fertilizing capacity. This study sought to determine whether red-light stimulation using a light emitting diode (LED) at 620-630 nm affects sperm motility and structure of motile subpopulations, sperm viability, mitochondrial activity, intracellular ATP levels, rate of O consumption and DNA integrity of horse spermatozoa. For this purpose, nine ejaculates were collected from nine different adult stallions. Upon collection, semen was diluted in Kenney extender, analyzed, its concentration was adjusted, and finally it was stimulated with red-light. In all cases, semen was packaged in 0.5-mL transparent straws, which were randomly divided into controls and 19 light-stimulation treatments; 6 consisted of a single exposure to red-light, and the other 13 involved irradiation with intervals of irradiation and darkness (light-dark-light). After irradiation, sperm motility was assessed using a Computerized Semen Analysis System (CASA). Flow cytometry was used to evaluate sperm viability, mitochondrial membrane potential and DNA fragmentation. Intracellular levels of ATP and O consumption rate were also determined. Specific red-light patterns were found to modify kinetics parameters (patterns: 4, 2-2-2, 3-3-3, 4-4-4, 5-1-5, and 5-5-5 min), the structure of motile sperm subpopulations (patterns: 2, 2-2-2, 3-3-3, and 4-1-4 min), mitochondrial membrane potential (patterns: 4, 3-3-3, 4-4-4, 5-1-5, 5-5-5, 15-5-15, and 15-15-15 min), intracellular ATP levels and the rate of O consumption (pattern: 4 min), without affecting sperm viability or DNA integrity. Since the increase in some kinematic parameters was concomitant with that of mitochondrial activity, intracellular ATP levels and O consumption rate, we suggest that the positive effect of light-irradiation on sperm motility is related to its impact upon mitochondrial activity. In conclusion, this study shows that red LED light stimulates motility and mitochondrial activity of horse sperm. Additional research is needed to address the impact of red-light irradiation on fertilizing ability and the mechanisms through which light exerts its effects

Effect of Exposure to Seminal Plasma Through Natural Mating in Cattle on Conceptus Length and Gene Expression

peer-reviewedA growing body of evidence suggests that paternal factors have an impact on offspring development. These studies have been mainly carried out in mice, where seminal plasma (SP) has been shown to regulate endometrial gene expression and impact embryo development and subsequent offspring health. In cattle, infusion of SP into the uterus also induces changes in endometrial gene expression, however, evidence for an effect of SP on early embryo development is lacking. In addition, during natural mating, the bull ejaculates in the vagina; hence, it is not clear whether any SP reaches the uterus in this species. Thus, the aim of the present study was to determine whether SP exposure leads to improved early embryo survival and developmental rates in cattle. To this end, Day 7 in vitro produced blastocysts were transferred to heifers (12–15 per heifer) previously mated to vasectomized bulls (n = 13 heifers) or left unmated (n = 12 heifers; control). At Day 14, heifers were slaughtered, and conceptuses were recovered to assess size, morphology and expression of candidate genes involved in different developmental pathways. Additionally, CL volume at Day 7, and weight and volume of CL at Day 14 were recorded. No effect of SP on CL volume and weight not on conceptus recovery rate was observed. However, filamentous conceptuses recovered from SP-exposed heifers were longer in comparison to the control group and differed in expression of CALM1, CITED1, DLD, HNRNPDL, PTGS2, and TGFB3. In conclusion, data indicate that female exposure to SP during natural mating can affect conceptus development in cattle. This is probably achieved through modulation of the female reproductive environment at the time of mating. Keywords: seminal plasma, embryo development, corpus luteu

Extracellular Reactive Oxygen Species (ROS) Production in Fresh Donkey Sperm Exposed to Reductive Stress, Oxidative Stress and NETosis

Altres ajuts: Generalitat de Catalunya 2017-SGR-1229Jenny shows a large endometrial reaction after semen influx to the uterus with a large amount of polymorphonuclear neutrophils (PMN) migrating into the uterine lumen. PMN act as a sperm selection mechanism through phagocytosis and NETosis (DNA extrudes and, together with proteins, trap spermatozoa). While a reduced percentage of spermatozoa are phagocytosed by PMN, most are found to be attached to neutrophil extracellular traps (NETs). This selection process together with sperm metabolism produces a large amount of reactive oxygen species (ROS) that influence the reproductive success. The present study aimed to determine the extracellular ROS production in both sperm and PMN. With this purpose, (1) donkey sperm were exposed to reductive and oxidative stresses, through adding different concentrations of reduced glutathione (GSH) and hydrogen peroxide (HO), respectively; and (2) PMN were subjected to NETosis in the presence of the whole semen, sperm, seminal plasma (SP) or other activators such as formyl-methionyl-leucyl-phenylalanine (FMLP). Extracellular ROS production (measured as HO levels) was determined with the Amplex ® Red Hydrogen Peroxide/Peroxidase Assay Kit. Donkey sperm showed more resilience to oxidative stress than to the reductive one, and GSH treatments led to greater HO extracellular production. Moreover, not only did SP appear to be the main inducer of NETosis in PMN, but it was also able to maintain the extracellular HO levels produced by sperm and NETosis

Novel insights into paternal factors influencing the maternal environment and embryo development

For many years, reproductive outcomes were considered as being solely determined by the genome of oocytes and sperm. Yet, in the last decades, many other female and male factors have also been found to be relevant. Focusing on the latter, paternal factors have been proposed to be capable of modulating multiple features of the reproductive process, such as sperm physiology, the maternal environment and, even, the offspring health. Considering that the molecular mechanisms underlying this paternal regulation are mostly unknown, the objective of the present Dissertation was to increase the current knowledge on the role played by seminal plasma (SP) and sperm on the reproductive success, using the bovine and porcine as animal models. Chapter 1 investigated the involvement of SP in the modulation of in vivo fertility and the potential pathways behind this regulation. The SP was found to be able to evoke changes in the female reproductive environment, with a positive effect on early embryo development. Next, the protein (particularly AKR1B1) and metabolite composition of SP was examined to address the potential molecular mechanisms able to modulate in vivo fertility. The results indicated that the positive influence of SP on in vivo fertility outcomes could be attributed to the modulation of the female reproductive tract and sperm physiology. On the other hand, Chapter 2 sought to determine to what extent sperm components, including proteins, metabolites and DNA integrity, can shape in vitro fertility outcomes, particularly oocyte fertilization and pre-implantation embryo development. Data showed that the AKR1B1 present in sperm is involved in the regulation of sperm fertilising ability. In addition, sperm metabolism is also able to influence early embryo development. One feasible explanation for such findings is that sperm AKR1B1 levels and metabolism could affect the production of reactive oxygen species, which are known to induce DNA breaks. For this reason, the last study investigated if DNA breaks might influence in vitro fertility outcomes. Interestingly, DNA fragmentation was identified to compromise embryo development, but not sperm fertilizing ability. In conclusion, the findings of this Dissertation supported that seminal factors can directly and indirectly modulate the maternal environment and influence embryo development. Further research should be focused on comprehensively determining the male-female-embryo cross-talk to better understand which factors underlie the reproductive success in mammalsDurant força temps, el paradigma dominant de la Biologia de la Reproducció ha considerat que l’èxit reproductiu depèn únicament del genoma dels oòcits i els espermatozoides. Tanmateix, en les últimes dècades, s’ha descrit la rellevància d’altres factors, tant femenins com masculins. Concretament, s’ha observat que aquests darrers són capaços de modular diferents aspectes essencials del procés reproductiu, com ara la fisiologia espermàtica, l’ambient matern i, fins i tot, la salut de la descendència. Tenint en compte que els mecanismes moleculars subjacents a aquesta regulació no es coneixen amb profunditat, l’objectiu d’aquesta Tesi Doctoral va ser determinar el paper del plasma seminal (PS) i els espermatozoides en l’èxit reproductiu, emprant les espècies bovina i porcina com a animals model. El Capítol 1 va investigar la implicació del PS en la modulació de la fertilitat in vivo i les vies moleculars relacionades amb aquesta regulació. Es va observar que el PS és capaç de provocar canvis en l'ambient reproductiu femení, la qual cosa es tradueix en un efecte positiu en el desenvolupament embrionari primerenc. Seguidament, es va examinar la composició del PS en termes de proteïnes (particularment AKR1B1) i metabòlits per tal de determinar si aquestes molècules poden alterar la fertilitat in vivo. Els resultats van indicar que la influència positiva d’aquestes molècules en els resultats de fertilitat in vivo podia atribuir-se a la modulació del tracte reproductor femení i la fisiologia de l'espermatozoide. D'altra banda, el Capítol 2 va investigar si els components de l'espermatozoide, incloent proteïnes, metabòlits i la integritat de l’ADN espermàtic, poden influir en la fertilitat in vitro, particularment en la fecundació de l'oòcit i el desenvolupament embrionari pre-implantacional. Es va observar que l’AKR1B1 present a l'espermatozoide està involucrada en la regulació de la seva capacitant fecundant. A més, també es va trobar que el metabolisme de l'espermatozoide influencia el desenvolupament embrionari primerenc. Amb tot plegat, es va suggerir que els nivells d'AKR1B1 de l'espermatozoide i el seu metabolisme podrien afectar la producció d’espècies reactives d'oxigen, que es coneix que indueixen trencaments de ADN. Per aquest motiu, l’últim estudi va investigar si la fragmentació de l’ADN pot afectar l’èxit de la fecundació in vitro. Es va observar que els danys a l’ADN espermàtic comprometen el desenvolupament embrionari però no la capacitat fecundant dels espermatozoides. En conclusió, els resultats d'aquesta Tesi Doctoral demostren que els factors paterns poden influir directament i indirecta tant en l'entorn matern com en la fecundació dels oòcits i el posterior desenvolupament de l'embrió. Per aquest motiu, els estudis futurs s'haurien de centrar en determinar exhaustivament la comunicació entre mascle-femella-embrió per entendre millor els factors subjacents a l'èxit reproductiu en els mamífersPrograma de Doctorat en Tecnologi

Cryopreservation and oxidative stress in porcine oocytes

Several vitrification protocols have been established for porcine oocytes so as to facilitate gene banking of female germplasm. Although live piglets have been successfully produced from pig oocytes vitrified at the germinal vesicle (GV) stage, the competence of vitrified oocytes to develop into the blastocyst stage is greatly compromised following cryopreservation. The focus of this review is to elucidate the impact of cryopreservation on the redox balance of pig oocytes, making special reference to the relevance of non-enzymatic and enzymatic antioxidant defences. Besides, the regulation of gene expression in response to oxidative stress is also considered. Finally, we discuss the effects of supplementing maturation and vitrification media with the exogenous nonenzymatic antioxidants that have hitherto yielded the most relevant results

Embryo development is impaired by sperm mitochondrial-derived ROS.

BACKGROUND: Basal energetic metabolism in sperm, particularly oxidative phosphorylation, is known to condition not only their oocyte fertilising ability, but also the subsequent embryo development. While the molecular pathways underlying these events still need to be elucidated, reactive oxygen species (ROS) could have a relevant role. We, therefore, aimed to describe the mechanisms through which mitochondrial activity can influence the first stages of embryo development. RESULTS: We first show that embryo development is tightly influenced by both intracellular ROS and mitochondrial activity. In addition, we depict that the inhibition of mitochondrial activity dramatically decreases intracellular ROS levels. Finally, we also demonstrate that the inhibition of mitochondrial respiration positively influences sperm DNA integrity, most likely because of the depletion of intracellular ROS formation. CONCLUSION: Collectively, the data presented in this work reveals that impairment of early embryo development may result from the accumulation of sperm DNA damage caused by mitochondrial-derived ROS