RESUMO
Sperm quality is defined as the sperm cell ability to successfully fertilize eggs and allow normal embryo developmentâ . Few studies explore sperm quality using aquatic invertebrates. Parhyale hawaiensis is a marine amphipod with a circumtropical distribution and considered a model for evolution, development, and ecotoxicological studies. We aimed to develop a methodology to collect sperm cells of P. hawaiensis and evaluate their viability and DNA damage (comet assay). We directly exposed the sperm cells to different mutagenic agents to optimize/develop the protocols. Then, as a proof of concept, we exposed the males to mutagenic compounds (EMS, benzo[a]pyrene (BaP), azo and anthraquinone dyes) at non-lethal concentrations verified by the proposed viability test and analyzed their sperm cells for DNA damage (comet assay). Organisms exposed to EMS presented a clear concentration response in the DNA damage response. We also showed that BaP was able to induce a statistically significant increase in DNA damage of the sperm cells. For the two dyes, although DNA damage increased, statistically differences were not observed. We believe we successfully developed a test to detect genotoxicity of chemicals in sperm cells using an invertebrate model. The protocol for sperm cell viability needs to be further explored with different chemicals to verify its utility as a toxicity endpoint. The developed genotoxicity test has the advantages to employ organisms that are easily cultivated in reduced space, use simple laboratory resources and reduced amount of material and reagents. Positive responses with this model could be used to disclose new germ cell mutagen candidates which could be further confirmed in vertebrates' systems.
Assuntos
Anfípodes , Sobrevivência Celular , Dano ao DNA , Espermatozoides , Poluentes Químicos da Água , Animais , Masculino , Anfípodes/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Mutagênicos/toxicidade , Ensaio CometaRESUMO
This study investigated the impact of various Ge132 (Bis-carboxyethyl germanium sesquioxide) concentrations on frozen bovine semen. Ejaculates from three bulls were pooled and divided into six groups, each one with different Ge132 concentrations (0, 500, and 1000 µg/mL) and each group was incubated in different conditions (33°C for 30 min (D: D0, D500, and D1000), and the other was immediately cooled to 4°C (R: R0-control; R500 and R1000)). Thawed semen was evaluated for sperm characteristics by CASA and flow cytometer. Results showed better motility in the immediate cooling group without Ge132 compared with high Ge132 concentrations. Values for total motility dropped after 5 and 60 min in groups with high Ge132 levels and some control groups. Linearity increased with 1000 µg/mL Ge132, while straightness differed between moments in multiple groups. Membrane integrity was higher in a control group and certain Ge132 groups. Lower O2 - generation occurred without Ge132. After oxidative stress induction, lipid peroxidation intensity increased with arachidonic acid, but D1000 had lower peroxidation than R0. Overall, Ge132 appears to have provided protection against PLM when subjected to oxidative stress, since even at high concentrations it maintained sperm metabolism.
Assuntos
Antioxidantes , Criopreservação , Preservação do Sêmen , Motilidade dos Espermatozoides , Espermatozoides , Animais , Masculino , Bovinos , Criopreservação/veterinária , Criopreservação/métodos , Preservação do Sêmen/veterinária , Preservação do Sêmen/métodos , Espermatozoides/efeitos dos fármacos , Antioxidantes/farmacologia , Motilidade dos Espermatozoides/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Crioprotetores/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Germânio/farmacologia , Sêmen/efeitos dos fármacos , Análise do Sêmen/veterináriaRESUMO
After ejaculation, mammalian sperm undergo a series of molecular events conducive to the acquisition of fertilizing competence. These events are collectively known as capacitation and involve acrosomal responsiveness and a vigorous sperm motility called hyperactivation. When mimicked in the laboratory, capacitating bovine sperm medium contains bicarbonate, calcium, albumin and heparin, among other components. In this study, we aimed at establishing a new capacitation protocol for bovine sperm, using calcium ionophore. Similar to our findings using mouse sperm, bovine sperm treated with Ca2+ ionophore A23187 were quickly immobilized. However, these sperm initiated capacitation after ionophore removal in fresh medium without heparin, and independent of the Protein Kinase A. When A23187-treated sperm were used on in vitro fertilization (IVF) procedures without heparin, eggs showed cleavage rates similar to standardized IVF protocols using heparin containg synthetic oviduct fluid (IVF-SOF). However, when A23187 pre-treated sperm were further used for inseminating eggs in complete IVF-SOF-heparin, a significantly higher percentage of embryo development was observed, suggesting a synergism between two different signaling pathways during bovine sperm capacitation. These results have the potential to improve current protocols for bovine IVF that could also be applied in other species of commercial interest.
Assuntos
Calcimicina , Ionóforos de Cálcio , Criopreservação , Fertilização in vitro , Preservação do Sêmen , Capacitação Espermática , Espermatozoides , Animais , Bovinos , Masculino , Ionóforos de Cálcio/farmacologia , Criopreservação/veterinária , Criopreservação/métodos , Fertilização in vitro/veterinária , Fertilização in vitro/métodos , Calcimicina/farmacologia , Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologia , Capacitação Espermática/efeitos dos fármacos , Preservação do Sêmen/veterinária , Preservação do Sêmen/métodos , Feminino , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/efeitos dos fármacosRESUMO
BACKGROUND: Exposure of humans and animals to heavy metals is increasing day-by-day; thus, lead even today remains of significant public health concern. According to CDC, blood lead reference value (BLRV) ranges from 3.5 µg/dl to 5 µg/dl in adults. Recently, almost 2.6% decline in male fertility per year has been reported but the cause is not well established. Lead (Pb2+) affects the size of testis, semen quality, and secretory functions of prostate. But the molecular mechanism(s) of lead toxicity in sperm cells is not clear. Thus, present study was undertaken to evaluate the adverse effects of lead acetate at environmentally relevant exposure levels (0.5, 5, 10 and 20 ppm) on functional and molecular dynamics of spermatozoa of bucks following in vitro exposure for 15 min and 3 h. RESULTS: Lead significantly decreased motility, viable count, and motion kinematic patterns of spermatozoa like curvilinear velocity, straight-line velocity, average path velocity, beat cross frequency and maximum amplitude of head lateral displacement even at 5 ppm concentration. Pb2+ modulated intracellular cAMP and Ca2+ levels in sperm cells through L-type calcium channels and induced spontaneous or premature acrosome reaction (AR) by increasing tyrosine phosphorylation of sperm proteins and downregulated mitochondrial transmembrane potential. Lead significantly increased DNA damage and apoptosis as well. Electron microscopy studies revealed Pb2+ -induced deleterious effects on plasma membrane of head and acrosome including collapsed cristae in mitochondria. CONCLUSIONS: Pb2+ not only mimics Ca2+ but also affects cellular targets involved in generation of cAMP, mitochondrial transmembrane potential, and ionic exchange. Lead seems to interact with Ca2+ channels because of charge similarity and probably enters the sperm cell through these channels and results in hyperpolarization. Our findings also indicate lead-induced TP and intracellular Ca2+ release in spermatozoa which in turn may be responsible for premature acrosome exocytosis which is essential feature of capacitation for fertilization. Thus, lead seems to reduce the fertilizing capacity of spermatozoa even at 0.5 ppm concentrations.
Assuntos
Reação Acrossômica , Acrossomo , Cálcio , Chumbo , Motilidade dos Espermatozoides , Espermatozoides , Masculino , Espermatozoides/efeitos dos fármacos , Cálcio/metabolismo , Motilidade dos Espermatozoides/efeitos dos fármacos , Animais , Acrossomo/efeitos dos fármacos , Chumbo/toxicidade , Reação Acrossômica/efeitos dos fármacos , AMP Cíclico/metabolismo , Bovinos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Análise do Sêmen , Dano ao DNA/efeitos dos fármacos , Compostos Organometálicos/toxicidade , Compostos Organometálicos/farmacologiaRESUMO
Although under appropriate laboratory conditions, sperm from different mammalian species can be capacitated in vitro, the optimal conditions for sperm capacitation in the stallion have been elusive. This study evaluated the effect of different capacitating inducers in Whitten and Tyrode media and assessed their impact on capacitation-related factors. Stallion sperm were incubated with different combinations of capacitating inducers at 38.5 °C in an air atmosphere. Sperm quality variables such as motility, mitochondrial membrane potential, and lipid peroxidation were assessed. Membrane fluidity and intracellular calcium levels were evaluated as early markers of capacitation, while tyrosine phosphorylation events and the sperm's ability to perform acrosomal exocytosis were used as late capacitation markers. Finally, these sperm were evaluated using a heterologous zona pellucida binding assay. The findings confirm that capacitating conditions evaluated increase intracellular calcium levels and membrane fluidity in both media. Similarly, including 2 or 3 inducers in both media increased tyrosine phosphorylation levels and acrosomal exocytosis after exposure to progesterone, confirming that stallion sperm incubated in these conditions shows cellular and molecular changes consistent with sperm capacitation. Furthermore, the zona pellucida binding assay confirmed the binding capacity of sperm incubated in capacitation conditions, a key step for stallion in vitro fertilization success. Further studies are needed to evaluate the effect of these conditions on in vitro fertilization in the horse.
Assuntos
Capacitação Espermática , Espermatozoides , Animais , Capacitação Espermática/efeitos dos fármacos , Masculino , Cavalos/fisiologia , Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologia , Cálcio/metabolismo , Zona Pelúcida/efeitos dos fármacos , Motilidade dos Espermatozoides/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , FosforilaçãoRESUMO
Superparamagnetic iron oxide nanoparticles (SPIONs) have gained significant attention in biomedical research due to their potential applications. However, little is known about their impact and toxicity on testicular cells. To address this issue, we conducted an in vitro study using primary mouse testicular cells, testis fragments, and sperm to investigate the cytotoxic effects of sodium citrate-coated SPIONs (Cit_SPIONs). Herein, we synthesized and physiochemically characterized the Cit_SPIONs and observed that the sodium citrate diminished the size and improved the stability of nanoparticles in solution during the experimental time. The sodium citrate (measured by thermogravimetry) was biocompatible with testicular cells at the used concentration (3%). Despite these favorable physicochemical properties, the in vitro experiments demonstrated the cytotoxicity of Cit_SPIONs, particularly towards testicular somatic cells and sperm cells. Transmission electron microscopy analysis confirmed that Leydig cells preferentially internalized Cit_SPIONs in the organotypic culture system, which resulted in alterations in their cytoplasmic size. Additionally, we found that Cit_SPIONs exposure had detrimental effects on various parameters of sperm cells, including motility, viability, DNA integrity, mitochondrial activity, lipid peroxidation (LPO), and ROS production. Our findings suggest that testicular somatic cells and sperm cells are highly sensitive and vulnerable to Cit_SPIONs and induced oxidative stress. This study emphasizes the potential toxicity of SPIONs, indicating significant threats to the male reproductive system. Our findings highlight the need for detailed development of iron oxide nanoparticles to enhance reproductive nanosafety.
Assuntos
Nanopartículas Magnéticas de Óxido de Ferro , Espermatozoides , Testículo , Masculino , Animais , Camundongos , Testículo/efeitos dos fármacos , Nanopartículas Magnéticas de Óxido de Ferro/toxicidade , Nanopartículas Magnéticas de Óxido de Ferro/química , Espermatozoides/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Citrato de Sódio , Células CultivadasRESUMO
To acquire the ability to fertilize the egg, mammalian spermatozoa must undergo a series of changes occurring within the highly synchronized and specialized environment of the female reproductive tract, collectively known as capacitation. In an attempt to replicate this process in vitro, various culture media for mouse sperm were formulated over the past decades, sharing a similar overall composition but differing mainly in ion concentrations and metabolic substrates. The widespread use of the different media to study the mechanisms of capacitation might hinder a comprehensive understanding of this process, as the medium could become a confounding variable in the analysis. In this context, the present side-by-side study compares the influence of four commonly used culture media (FD, HTF and two TYH versions) on mouse sperm capacitation. We evaluated the induction of protein kinase A phosphorylation pathway, motility, hyperactivation and acrosome reaction. Additionally, in vitro fertilization and embryo development were also assessed. By analyzing these outcomes in two mouse colonies with different reproductive performance, our study provides critical insights to improve the global understanding of sperm function. The results obtained highlight the importance of considering variations in medium composition, and their potential implications for the future interpretation of results.
Assuntos
Reação Acrossômica , Meios de Cultura , Fertilização in vitro , Capacitação Espermática , Espermatozoides , Animais , Capacitação Espermática/efeitos dos fármacos , Masculino , Camundongos , Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologia , Espermatozoides/metabolismo , Fertilização in vitro/métodos , Feminino , Reação Acrossômica/efeitos dos fármacos , Motilidade dos Espermatozoides/efeitos dos fármacos , Fosforilação , Fertilização , Desenvolvimento Embrionário/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismoRESUMO
OBJECTIVE: Clinacanthus nutans (C. nutans) is a medicinal herb that most people with diabetes have historically taken. It's a diet high in antioxidants, which are supposed to help people live longer and be healthier. It is the first study to suggest using C. nutans to enhance the quality of sperm in male mice given a streptozotocin (STZ) injection. METHODS: Sixty mice were divided into two groups at the age of four weeks: group one was fed a regular diet (n=10), while group two was administered a high-fat diet (n=50) for eight weeks to develop obesity. Obese mice were given 100mg/kg of STZ to produce hyperglycemia with a 20% mortality rate. Then, 40 hyperglycemic mice were separated into two groups: STZ (n=10) and sample (n=30). The treatment groups were administered a methanolic extract of C. nutans leaves by gavage at doses of 150, 300, and 500mg/kg of body weight (n=10) for 4 weeks. RESULTS: In contrast to the STZ group, there was a substantial (p=0.001) drop in serum blood glucose and total sperm abnormalities in mice at varying doses. Catalase, glutathione s-transferase (GST), and total antioxidant capacity significantly (p=0.001) increased in the STZ mice group at varying doses, but malondialdehyde was reduced. In comparison to STZ mice, testosterone and luteinizing hormone (LH) levels improved in mice treated with extracts of C. nutans at various doses. For all of the following dependent variables, extraction of the leaf at higher concentrations of 500 milligrams/kilogram has better efficacy than 300 and 150 mg/kg after 4 weeks of treatment. CONCLUSIONS: The research and development of new natural agents to combat oxidative stress-related diseases have sparked a lot of interest. As a result, the potential leaf extract of C. nutans contains anti-hyperglycemic compounds and improves the quality of sperm in male mice.
Assuntos
Acanthaceae , Antioxidantes , Diabetes Mellitus Experimental , Extratos Vegetais , Folhas de Planta , Espermatozoides , Animais , Masculino , Camundongos , Extratos Vegetais/farmacologia , Antioxidantes/farmacologia , Espermatozoides/efeitos dos fármacos , Folhas de Planta/química , Acanthaceae/química , Diabetes Mellitus Experimental/tratamento farmacológico , Estreptozocina , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Análise do Sêmen , Estresse Oxidativo/efeitos dos fármacosRESUMO
Intracytoplasmic sperm injection (ICSI) in horses is currently employed for clinical and commercial uses, but the protocol could be optimized to improve its efficiency. We have hypothesized that destabilization of plasma and acrosomal membranes prior to injection would positively impact the developmental potential of equine zygotes generated by ICSI. This study evaluated effects of the sperm treatment with lysolecithin on plasma and acrosomal membranes and on oocyte activation ability, initially following heterologous ICSI on bovine oocytes and subsequently employing equine oocytes. The effects of the lysolecithin -treatment on the efficiency of conventional and piezo-assisted equine ICSI were evaluated. To do this, the equine sperm were treated with different concentrations of lysolecithin and the sperm plasma membrane, acrosome and DNA integrity were evaluated by flow cytometry. The results showed that a lysolecithin concentration of 0.08 % destabilized the membranes of all sperm and affected DNA integrity within the range described for the species (8-30 %). In addition, the heterologous ICSI assay showed that lysolecithin treatment was detrimental to the sperm's ability to activate the oocyte, therefore, chemical oocyte activation was used after equine ICSI after injection with lysolecithin -treated sperm. This group showed similar developmental rate to the control group with and without exogenous activation. In conclusion, lysolecithin pre-treatment is not necessary when using ICSI to produce equine embryos in vitro. The results from the current study provide additional insight regarding the factors impacting ICSI in horses.
Assuntos
Lisofosfatidilcolinas , Injeções de Esperma Intracitoplásmicas , Espermatozoides , Animais , Cavalos , Injeções de Esperma Intracitoplásmicas/veterinária , Injeções de Esperma Intracitoplásmicas/métodos , Masculino , Lisofosfatidilcolinas/farmacologia , Espermatozoides/efeitos dos fármacos , Feminino , Oócitos/efeitos dos fármacosRESUMO
The aim of this study was to assess the addition of 2% sodium caseinate in a commercial egg yolk-based medium in frozen ovine semen. Eight Dorper males were used for the study. The ejaculate was divided into two portions and frozen without (G1) or with the addition of 2% sodium caseinate (G2). Kinetic parameters were evaluated using CASA (computer-assisted sperm analysis), and membrane and acrosome integrity as well as oxidative stress were assessed using flow cytometry. After thawing, a thermoresistance test was conducted at time points T0 and T90. For the fertility test, 100 ewes were inseminated with semen from two rams selected based on in vitro parameters, one with good post-thaw quality (+70% total motility) and the other with low post-thaw quality (-55% total motility). For the fertility test, the females were divided into 4 groups for insemination: low-quality ram without caseinate (GBS = 25) and with caseinate (GBC = 25), and high-quality ram without caseinate (GAS = 25) and with caseinate (GAC = 25). Regarding the results of sperm kinetics, there was a statistically significant difference in the parameters of average path velocity (VAP) and curvilinear velocity (VCL) between the group frozen with BotuBov and the group with added caseinate. At time point T90, straight-line velocity maintained a trend (p < .06), with BotuBov® (BB group) being superior to caseinate this time, and in the linearity parameter, caseinate was superior to BotuBov®. Flow cytometry analysis showed no difference between any of the evaluated tests. In the fertility test, there was no statistically significant difference in the pregnancy rate between the BotuBOV® group (23%, 11/48) and the sodium caseinate group (BC group) (33%, 17/52), and no differences were observed in the male versus diluent interaction (p = .70). In conclusion, sodium caseinate supplementation did not influence sperm kinetic parameters and the fertility of sheep.