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To unravel the toxic mechanism of phthalate ester plasticizer endocrine disruptor in spermatozoa, we examined the effect of dibutyl phthalate (DBP) on the stability and inhibitory phosphorylation of glycogen synthase kinase 3α (GSK3α), a protein kinase crucial for sperm motility in mice. In DBP-treated spermatozoa, reactive oxygen species (ROS) and lipid peroxide were significantly increased. In computer-assisted sperm analysis, DBP at concentrations of 10 – 100 μg/mL significantly decreased total motility and progressive motility of spermatozoa. On western blots, DBP decreased p-GSK3α(Ser21) and increased p-GSK3α(Tyr279) in spermatozoa. Similarly, hydrogen peroxide decreased p-GSK3α(Ser21) but not p-GSK3α(Tyr279) in spermatozoa. Immunofluorescent labeling demonstrated that DBP markedly decreased immunoreactivities of GSK3α and p-GSK3α(Ser21) but increased immunoreactivity of p-GSK3α(Tyr279) in spermatozoa. DBP at a concentration of 100 μg/mL significantly increased phosphatase activity in spermatozoa. Calyculin A, a protein phosphatase 1 and 2 A inhibitor, markedly increased p-GSK3α(Ser21) and sperm motility and attenuated a DBP-induced decrease of p-GSK3α(Ser21) and sperm motility. On western blot, 1–100 μg/mL DBP decreased GSK3α in spermatozoa. On immunoprecipitation western blot, DBP at 10 - 100 μg/mL increased polyubiquitinated sperm proteins including GSK3α. The MG115, proteasome inhibitor attenuated degradation of GSK3α in DBP-treated spermatozoa. Hydrogen peroxide at 10 μM increased polyubiquitinated sperm proteins, suggesting that DBP may increase ubiquitination of GSK3α via ROS induction. Together, DBP may decrease the cellular amount of GSK3α through the ubiquitin-proteasome pathway and p-GSK3α(Ser21) through ROS generation and activation of protein phosphatases, impairing sperm motility.

Background: We previously demonstrated how mouse spermatozoa can be efficiently stored for two years in a −80°C freezer, maintaining their ability to fertilize mouse eggs. Objectives: The main objective here was to evaluate the effects of five years at −80°C compared to liquid nitrogen storage (LN2, control condition) on mouse sperm viability, physiological parameters, and fertilization capacity. Materials and methods: Three different strains were used: C57BL/6N, C57BL/6J and CD1. Flow cytometry experiments were performed to analyze sperm viability (SYBR‐14 + Propidium Iodide +Hoechst33342), the intracellular calcium concentration (Fluo 3‐AM), the membrane lipid disorder (Merocyanine 540), and the mitochondrial activity (MitoTracker Red) in live spermatozoa. The in vitro fertilization (IVF) was used to evaluate the sperm fertilizing ability. Results: Flow cytometry analysis showed that the percentage of live cells are reduced in B6N and B6J, but not in CD1 mice. However, in the live population no differences in terms of intracellular calcium concentration, membrane lipid disorder, and mitochondrial activity were reported when comparing both biobanking methods. Spermatozoa stored at −80°C for 5 years successfully fertilized the eggs and developed mouse embryo normally both in culture and in vivo, generating live pups with no differences compared to control samples stored in LN2. Discussion: Long‐term mouse sperm storage at −80°C (five years) could be considered an ideal alternative to the most common LN2 approach, giving economical and logistic advantages. Moreover, the precise information originated from the flow cytometry analysis stands up this technique as an optimal strategy to evaluate the sperm quality and ranking. Conclusion: It is demonstrated here the possibility to store mouse spermatozoa for up to five years in a −80°C freezer with no significant differences compared to the storage in LN2 in terms of fertilizing ability, sperm viability, intracellular calcium concentration, membrane lipid disorder, and mitochondrial activity. [ABSTRACT FROM AUTHOR]

During the latest years, human infertility worsened all over the world and is nowadays reputed as a global public health issue. As a consequence, the adoption of Assisted Reproductive Technologies (ARTs) such as In Vitro Fertilization (IVF) is undergoing an impressive increase. In this context, one of the most promising strategies is the innovative adoption of extra-physiological materials for advanced sperm preparation methods. Here, by using a murine model, the addition of Graphene Oxide (GO) at a specific concentration has demonstrated to increase the spermatozoa fertilizing ability in an IVF assay, finding that 0.5 μg/ml GO addition to sperm suspensions before IVF is able to increase both the number of fertilized oocytes and embryos created with a healthy offspring given by Embryo Transplantation (ET). In addition, GO treatment has been found more effective than that carried out with methyl-β-cyclodextrin, which represents the gold standard in promoting in vitro fertility of mice spermatozoa. Subsequent biochemical characterization of its interaction with male gametes has been additionally performed. As a result, it was found that GO exerts its positive effect by extracting cholesterol from membranes, without affecting the integrity of microdomains and thus preserving the sperm functions. In conclusion, GO improves IVF outcomes in vitro and in vivo, defining new perspectives for innovative strategies in the treatment of human infertility.

To unravel the toxic mechanism of phthalate ester plasticizer endocrine disruptor in spermatozoa, we examined the effect of dibutyl phthalate (DBP) on the stability and inhibitory phosphorylation of glycogen synthase kinase 3α (GSK3α), a protein kinase crucial for sperm motility in mice. In DBP-treated spermatozoa, reactive oxygen species (ROS) and lipid peroxide were significantly increased. In computer-assisted sperm analysis, DBP at concentrations of 10 – 100 μg/mL significantly decreased total motility and progressive motility of spermatozoa. On western blots, DBP decreased p-GSK3α(Ser21) and increased p-GSK3α(Tyr279) in spermatozoa. Similarly, hydrogen peroxide decreased p-GSK3α(Ser21) but not p-GSK3α(Tyr279) in spermatozoa. Immunofluorescent labeling demonstrated that DBP markedly decreased immunoreactivities of GSK3α and p-GSK3α(Ser21) but increased immunoreactivity of p-GSK3α(Tyr279) in spermatozoa. DBP at a concentration of 100 μg/mL significantly increased phosphatase activity in spermatozoa. Calyculin A, a protein phosphatase 1 and 2 A inhibitor, markedly increased p-GSK3α(Ser21) and sperm motility and attenuated a DBP-induced decrease of p-GSK3α(Ser21) and sperm motility. On western blot, 1–100 μg/mL DBP decreased GSK3α in spermatozoa. On immunoprecipitation western blot, DBP at 10 - 100 μg/mL increased polyubiquitinated sperm proteins including GSK3α. The MG115, proteasome inhibitor attenuated degradation of GSK3α in DBP-treated spermatozoa. Hydrogen peroxide at 10 μM increased polyubiquitinated sperm proteins, suggesting that DBP may increase ubiquitination of GSK3α via ROS induction. Together, DBP may decrease the cellular amount of GSK3α through the ubiquitin-proteasome pathway and p-GSK3α(Ser21) through ROS generation and activation of protein phosphatases, impairing sperm motility. [Display omitted] ● p-GSK3α(Ser21) decreased and p-GSK3α(Tyr279) increased during motility activation. ● DBP decreased p-GSK3α(Ser21) and motility of sperm but increased p-GSK3α(Tyr279). ● DBP increased phosphatase activity, decreasing p-GSK3α(Ser21) in spermatozoa. ● DBP increased degradation of GSK3α via the ubiquitin proteasome pathway. ● Disruption of p-GSK3α(Ser21) is pivotal toxic mechanism of DBP affecting motility. [ABSTRACT FROM AUTHOR]

Background: Reproductive toxicity of lipopolysaccharide (LPS) on spermatozoa is well established. Objective: The aim of the present study was to show the potential benefits of alpha lipoic acid (ALA) as a strong antioxidant in alleviating the reproductive toxicity of LPS. Materials and methods: Sperm cells and cumulus–oocyte complexes (COCs) were collected from healthy NMRI mice (body weights ranged from 25 to 35 g, 100 females and 200 males). Sperm cells were treated with varying doses of ALA (0.01, 0.02, and 0.04 mm) and 0.01 μg/mL of LPS for 4 h. The quality of spermatozoa (ROS production, DNA fragmentation, and spontaneous acrosome reaction), sperm fertilizability, and the consequent developmental competence of oocytes inseminated with ALA/LPS‐treated spermatozoa were recorded. Results: The results showed that 0.04 mm of ALA abrogated LPS‐reduced sperm motility, viability, ROS production, spontaneous acrosome reaction, fertilizability, and developmental competence. In addition, 0.04 mm ALA significantly reverted the negative effects of LPS on inner cell mass (ICM) cell counts, total cell number (TCM), and ratio between ICM and TCM. Discussion: Our data showed that ALA significantly could abrogate the negative effects of LPS on sperm quality and oocyte developmental competence. Therefore, ALA had the capacity for protecting sperm cells from LPS‐induced damage and ensured fertilization and developmental competency. Conclusion: These in vitro findings suggested a therapeutic role for ALA in reducing the negative effects of LPS on spermatozoa and early embryonic development. [ABSTRACT FROM AUTHOR]

Abstract Present study aimed to determine to what extent freeze-dried spermatozoa were able to withstand high-temperature conditions: transient increase in storage temperature and long-term exposure to room temperature. Mouse spermatozoa were freeze-dried in EGTA/Tris-HCl buffered solution alkalinized using KOH (K-ETBS, pH 7.7), and then stored for up to 7 months at 4 °C or 25 °C. After 2 months' storage, some of the 4°C-stored spermatozoa were exposed to 40 °C for 1 week or 1 month, then again stored at 4 °C for the remaining storage period. Following storage, rehydrated spermatozoa were injected into mouse oocytes. The resulting zygotes were assessed for chromosome damage, in vitro development up to the blastocyst stage, and post-implantation development to normal fetuses on day 18 of gestation. In storage at 4 °C, one-week exposure to 40 °C had no adverse effect on the chromosome integrity and developmental competence compared to non-exposure to 40 °C (continuous storage at 4 °C). In contrast, one-month exposure to 40 °C caused an increasing level of chromosome damage (36%, P < 0.05) and reduced frequencies of blastocysts (54%, P < 0.05) and normal fetuses (36%, P < 0.05) compared to the frequencies obtained by continuous storage at 4 °C (15%, 82% and 52%, respectively). Storage at 25 °C resulted in accumulation of chromosome damage (27%, P < 0.05), leading to decreased blastocyst formation (63%, P < 0.05). But, the frequency of normal fetus (44%) was not significantly different from that obtained by continuous storage at 4 °C. Consequently, mouse spermatozoa freeze-dried in K-ETBS withstood temporary exposure to 40 °C for 1 week. Chromosome damage accumulated in spermatozoa during storage at 25 °C. [ABSTRACT FROM AUTHOR]

The mouse is widely used for biomedical research and an increasing number of genetically altered models are currently generated, therefore centralized repositories are essentials to secure the important mouse strains that have been developed. We have previously reported that spermatozoa of wild type and mutant strains frozen using standard laboratory protocols can be transported in dry ice (−79 °C) for 7 days and safely stored in a −80 °C freezer for up to two years. The objective of this new study was to compare the effects of the freezing techniques using LN 2 or -80 °C freezer on fertility of frozen-thawed mouse spermatozoa. After thawing, sperm fertility was comparable (P > 0,05) between the LN 2 and the −80 °C samples for at least 1 year. Furthermore, we showed that it is possible to freeze and store mouse semen directly at −80 °C and eventually transfer it to LN 2 irrespective of storage time. This study is relevant because it shows for the first time that mouse spermatozoa can be efficiently frozen and stored at −80 °C with no use of liquid nitrogen for a long period of time. A new, simple, efficient and flexible, liquid nitrogen free, method was developed for freezing and maintaining spermatozoa of wild type and mutant C57BL/6N lines. Lines on this genetic background are used in collaborative research infrastructures for systematic phenotyping, e.g. the International Mouse Phenotyping Consortium (IMPC) and therefore largely cryopreserved in repositories like EMMA/Infrafrontier. The importance of this finding will be especially useful for small laboratories with no or limited access to liquid nitrogen and for laboratories generating many mouse mutant lines by CRISPR/Cas9 who do not want to saturate the limited space of a LN 2 tank, using a more accessible −80 °C freezer. This study underlines, once more, that mouse spermatozoa are very resistant and can be frozen, transported, shared and stored at −80 °C for a long time without a significant loss of viability. This new approach simplifies the freezing process and facilitates the long term storage of mouse spermatozoa at −80 °C, always allowing the transfer to LN 2 for indefinite storage without noticeable detrimental effects. [ABSTRACT FROM AUTHOR]

In order to protect sperm chromosomes against degradation when they are being stored for relatively high temperatures, we investigated the optimal pH of the freeze-drying solution, EGTA/Tris-HCl buffered solution alkalinized by NaOH (Na-ETBS) or KOH (K-ETBS). Mouse spermatozoa suspended in Na-ETBS or K-ETBS were freeze-dried at pH 5.0–8.4 and stored at 4 °C or 50 °C for 3 days. Some freeze-dried samples were stored at 25 °C for 3 days or 1 month. After storage, samples injected into oocytes using intracytoplasmic sperm injection were assessed for chromosome damage in resulting zygotes. Irrespective of freeze-drying solutions and storage temperatures, almost all the zygotes (97–100%) produced using the spermatozoa freeze-dried at pH 5.0 had structural chromosome aberrations of sperm origin. When freeze-drying was conducted at pH 8.0–8.4 using Na-ETBS, the incidence of chromosome damage decreased to 14–17% in 4 °C storage and 24–26% in 50 °C storage. When freeze-dried in K-ETBS, the lowest level of chromosome damage occurred at pH levels of 7.7–8.4 at 4 °C storage (13–15%) and at pH 7.7–8.0 at 50 °C storage (16–23%). Spermatozoa freeze-dried in Na-ETBS at pH 8.2 and K-ETBS at pH 7.7 showed no significant increase in chromosome damage during 25 °C storage from 3 days to 1 month (11%–20% in Na-ETBS; 13%–18% in K-ETBS). Thus, use of the solutions optimized for short-term storage at high temperature (50 °C, 3 days) permits prolonged storage (1 month) of freeze-dried spermatozoa at room temperature. [ABSTRACT FROM AUTHOR]

Due to their catalytic and oxidative properties, cerium dioxide nanoparticles (CeO2NPs) are widely used as diesel additive or as promising therapy in cancerology; yet, scarce data are available on their toxicity, and none on their reproductive toxicity. We showed a significant decrease of fertilization rate, assessed on 1272 oocytes, during in vitro fertilization (IVF) carried out in culture medium containing CeO2NP at very low concentration (0.01 mg.l−1). We also showed significant DNA damage induced in vitro by CeO2NP on mouse spermatozoa and oocytes at 0.01 mg.l−1 using Comet assay. Transmission Electron Microscopy did not detect any nanoparticles in the IVF samples at 0.01 mg.l−1, but showed, at high concentration (100 mg.l−1), their endocytosis by the cumulus cells surrounding oocytes and their accumulation along spermatozoa plasma membranes and oocytes zona pellucida. We did not observe any nanoparticles in the cytoplasm of spermatozoa, oocytes or embryos. This study demonstrates for the first time the impact of CeO2NP on in vitro fertilization, as well as their genotoxicity on mouse spermatozoa and oocytes, at low nanoparticle concentration exposure. Decreased fertilization rates may result from: (1) CeO2NP's genotoxicity on gametes; (2) a mechanical effect, disrupting gamete interaction and (3) oxidative stress induced by CeO2NP. These results add new and important insights with regard to the reproductive toxicity of nanomaterials requesting urgent evaluation, and support several publications on metal nanoparticles reprotoxicity. Our data highlight the need for in vivo studies after low-dose exposure. [ABSTRACT FROM AUTHOR]

Abstract: This study examined a method for analyzing the count, motility, and morphology of mouse epididymal sperm, optimizing the diluent, incubation time, sample concentration, and temperature, using a particle counter (CDA-500) to count and size sperm and a sperm quality analyzer (SQA-IIC) to measure sperm motility, quantified as the sperm motility index (SMI). The optimal conditions consisted of a 30-min incubation in D-MEM (Dulbecco''s modified Eagle''s medium; considering cost and availability) at 37°C, with 5×106 cellsmL−1 in the original solution. Furthermore, the influence of formalin fixation, and the correlation between the automated counter and a manual method were investigated. The sample fixation had no marked effect on the sperm count or morphology assessment. A linear correlation was observed between the manual and automated methods (y =0.920x +0.276; r 2 =0.571; p <0.001; range: (3–6)×106). The suitability of the proposed method was confirmed using spermatozoa prepared from mice treated with the reproductive toxin diethylstilbestrol (DES). Using sperm from the cauda epididymidis on one side per mouse, we confirmed that measurement of these sperm parameters using the two devices was simple, rapid, inexpensive, and reproducible. [Copyright &y& Elsevier]

The functional integrity of the cell membrane of the mice sperm after daidzein administration intraperitoneal was assessed by studying the swelling reaction of the sperm when suspended in distilled water. The results obtained in water test indicate that daidzein, one of the phytoestrogens, decreases the number of normal and live sperm (swollen) in the samples obtaining from vas deferens. This effect was statistically significant in the mice after 7 and 14 days of daidzein administration. In mice treated with daidzein the number of dead (unswollen) sperm was increased, especially in animals after 14 days of experiment. [ABSTRACT FROM AUTHOR]

The contra gestational effects of norethisterone and its main metabolites, 5 α-NET and 3 β5 α-NET, has been demonstrated in several species. However, the focus has been mainly on their effects in the uterus. We previously reported that 5 α-NET inhibits the progesterone-induced AR in pig spermatozoa and induces severe morphological damage to fertilized mouse oocytes. In the present study, we analysed the effects of these compounds on the fertilization process in vitro. Oocytes and spermatozoa were obtained from Balb/c female and C57BL/6J male mice, respectively. Both, the AR assays and the fertilization experiments were performed under different steroid treatment schemes using progesterone as a control. Results showed that norethisterone induced the AR, while 5 α-NET reduced the percentage of spermatozoa that had undergone progesterone-induced AR. Both 17 β-estradiol and 3 β5 α-NET induced the AR in a considerably lower percentage of spermatozoa than progesterone. In addition, when 5 α-NET was added to the medium simultaneously with progesterone at the moment of fertilization, the percentage of fertilized oocytes (two-cell stage) decreased by as much as 77% as compared with the control progesterone-treated group. All results suggest that these compounds can have important effects on the fertilization process. [ABSTRACT FROM AUTHOR]

The cytogenetic effects of (-)-epigallocatechin gallate (EGCG) on mouse spermatozoa were studied in vitro using an intracytoplasmic sperm injection (ICSI) technique. Spermatozoa were collected by the swim-up method and treated with EGCG at 1μM and 10μM. When motile, EGCG-treated spermatozoa were injected into oocytes, structural chromosome aberrations (SCAs) at the first cleavage metaphase did not increase significantly. However, a majority of immotile spermatozoa treated with 10μM EGCG had the following abnormalities: pronuclear arrest (11% of activated oocytes), degenerated sperm chromatin (chromosome) mass (30% of activated oocytes) and occurrence of structural chromosome aberrations (57% of analyzed metaphases). The incidence of these abnormalities suggests that immotile spermatozoa were susceptible to EGCG, and that the damage of sperm chromatin was accelerated in immotile spermatozoa by 10μM EGCG treatment. [Copyright &y& Elsevier]

Potential methods for cryopreservation of mouse spermatozoa are freeze-drying, desiccation, and suspension in EGTA Tris–HCl buffered solution (ETBS: 50 mM NaCl, 50 mM EGTA, and 10 mM Tris–HCl). To determine the duration that mouse spermatozoa suspended in ETBS-based solutions could retain their normal characteristics without freezing, spermatozoa collected from the cauda epididymis were suspended in ETBS or in ETBS supplemented with the antioxidants, dimethyl sulfoxide (DMSO), or dl-α-tocopherol acetate (Vitamin E acetate; VEA) diluted in DMSO, then held at ambient temperature (22–24 °C) for up to 9 days. When oocytes were injected with spermatozoa preserved in ETBS alone, activation rates of oocytes and chromosome integrity at the first cleavage metaphase decreased at 1 day (P<0.001) and 2–4 days (P<0.01) following treatment. When oocytes were injected with spermatozoa preserved in ETBS supplemented with DMSO or VEA/DMSO, chromosome integrity did not decrease significantly (through 9 days of preservation). Although DMSO maintained sperm chromosome integrity more effectively than VEA/DMSO up to 2–4 days (91 and 67%, normal karyotypes in DMSO and VEA/DMSO, respectively), VEA/DMSO helped to maintain the ability of spermatozoa to activate oocytes, but did not enhance the maintenance of sperm chromosome integrity. These results suggested that deterioration of spermatozoa preserved in ETBS alone was delayed by supplementation with antioxidants. [Copyright &y& Elsevier]

In the present study the effect of sodium nitroprusside, a nitric oxide releaser, on mouse sperm motion behavior was examined. The number of sperm migrating in experimental plate to medium containing sodium nitroprusside in concentration 50 nM was significantly higher than the number migrating to control. This effect was observed after 3 and 5 h of incubation, and it was not time dependent. The presence of sodium nitroprusside in higher concentrations did not cause any visible changes or decrease the migration of sperm. These effects were interpreted as chemokinesis but not chemotaxis of sperm induced by influence of sodium nitroprusside on sperm metabolism and motility. [ABSTRACT FROM AUTHOR]

The aim of this work was to evaluate the period of time required for the induction of changes in motility of mouse spermatozoa in response to factors from the microenvironment of oocytes. To determine the effects of the latency time, the period of preincubation before contact with the oocyte product(s), sperm samples were incubated for 15 or 90 min and then exposed to either Dulbecco's Modified Eagle's Medium (DMEM) or to a crude extract of superovulated oocytes (CE). The assays were performed in a Zigmond chamber by filling one compartment with either DMEM or CE, and the other compartment with the sperm suspension. A videomicroscopy system was used for tracking the spermatozoa. Sperm motility analysis was assessed using a semiautomatic objective method, and the following parameters were determined: (1) dynamic parameters: curvilinear velocity, linear velocity and linearity; (2) progressive motility: percentage of spermatozoa showing either circular or linear patterns of movement; and (3) directional motility: percentage of spermatozoa that moved towards either the DMEM or the CE. The results of this work showed that when the spermatozoa contacted soluble factors in CE after only 15 min of previous incubation, there was a significant increase in their dynamic parameters, change in their progressive motility, and induction of directional movement of the spermatozoa towards the CE components, while a longer period of preincubation did not significantly modify these effects. On the other hand, in the presence of culture medium (with or without addition of bovine serum albumin), the spermatozoa needed a more extended period of incubation to significantly increase their dynamic parameters and to modify their progressive motility, while maintaining a random direction of movement. [ABSTRACT FROM AUTHOR]