RESUMEN
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.
Asunto(s)
Reacción Acrosómica , Acrosoma , Calcio , Plomo , Motilidad Espermática , Espermatozoides , Masculino , Espermatozoides/efectos de los fármacos , Calcio/metabolismo , Motilidad Espermática/efectos de los fármacos , Animales , Acrosoma/efectos de los fármacos , Plomo/toxicidad , Reacción Acrosómica/efectos de los fármacos , AMP Cíclico/metabolismo , Bovinos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Análisis de Semen , Daño del ADN/efectos de los fármacos , Compuestos Organometálicos/toxicidad , Compuestos Organometálicos/farmacologíaRESUMEN
Follicular ovarian cysts (FOCs) are characterized by follicles in the ovaries that are >20 mm in diameter and persist for >10 days without the corpus luteum, leading to anovulation, dysregulation of folliculogenesis and subfertility in humans and livestock species. Despite their clinical significance, the precise impact of FOCs on oocyte reserve, maturation, and quality still needs to be explored. While FOCs are observed in both human and livestock populations, they are notably prevalent in livestock species. Consequently, livestock species serve as valuable models for investigating the molecular intricacies of FOCs. Thus, in this study, using goat FOCs, we performed integrated proteomic, metabolomic and functional analyses to demonstrate that oocyte maturation is hampered due to increased reactive oxygen species (ROS) in FOCs follicular fluid (FF) via downregulation of glutathione peroxidase (GPX1), a critical antioxidant seleno enzyme required to negate oxidative stress. Notably, GPX1 reduction was positively correlated with the FF's decline of free selenium and selenocysteine metabolic enzymes, O-phosphoryl-tRNA (Sec) selenium transferase (SEPSECS) and selenocysteine lyase (SCLY) levels. Adding GPX1, selenocysteine, or selenium to the culture media rescued the oocyte maturation abnormalities caused by FOCs FF by down-regulating the ROS. Additionally, we demonstrate that substituting GPX1 regulator, Insulin-like growth factor-I (IGF-1) in the in vitro maturation media improved the oocyte maturation in the cystic FF by down-regulating the ROS activity via suppressing Non-sense-mediated decay (NMD) of GPX1. In contrast, inhibition of IGF-1R and the target of rapamycin complex 1 (mTORC1) hampered the oocyte maturation via NMD up-regulation. These findings imply that the GPX1 regulation via selenocysteine metabolism and the IGF-1-mediated NMD may be critical for the redox homeostasis of FF. We propose that GPX1 enhancers hold promise as therapeutics for enhancing the competence of FOCs oocytes. However, further in vivo studies are necessary to validate these findings observed in vitro.
Asunto(s)
Líquido Folicular , Glutatión Peroxidasa GPX1 , Homeostasis , Factor I del Crecimiento Similar a la Insulina , Quistes Ováricos , Oxidación-Reducción , Selenocisteína , Femenino , Líquido Folicular/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Animales , Quistes Ováricos/metabolismo , Quistes Ováricos/patología , Selenocisteína/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Cabras , Estrés Oxidativo , Glutatión Peroxidasa/metabolismo , Glutatión Peroxidasa/genética , Oocitos/metabolismo , Humanos , Folículo Ovárico/metabolismo , Folículo Ovárico/patología , Proteómica/métodosRESUMEN
Earlier we have reported mercury-induced alterations in functional dynamics of buck spermatozoa through free radicals-mediated oxidative stress and spontaneous acrosome reaction. Based on our earlier findings, we aimed to investigate the effect of mercury exposure on motility, kinematic patterns, DNA damage, apoptosis and ultra-structural alterations in goat spermatozoa following in vitro exposure to different concentrations (0.031-1.25 µg/ml) of mercuric chloride for 15 min and 3 h. Following exposure of sperm cells to 0.031 µg/ml of mercuric chloride for 3 h, livability and motility of sperms was significantly reduced along with altered kinematic patterns, significant increase in per cent necrotic sperm cells and number of cells showing DNA damage; and this effect was dose- and time-dependent. Contrary to up-regulation of Bax gene after 3 h in control group, there was significant increase in expression of Bcl-2 in mercury-treated groups. Transmission electron microscopy studies revealed rifts and nicks in plasma and acrosomal membrane, mitochondrial sheath, and collapsed mitochondria with loss of helical organization of mitochondria in the middle piece of spermatozoa. Our findings evidently suggest that mercury induces necrosis instead of apoptosis and targets the membrane, acrosome, mid piece of sperms; and the damage to mitochondria seems to be responsible for alterations in functional and kinematic attributes of spermatozoa.
Asunto(s)
Mercurio/toxicidad , Mitocondrias/patología , Membranas Mitocondriales/patología , Capacitación Espermática/efectos de los fármacos , Motilidad Espermática/efectos de los fármacos , Espermatozoides/patología , Animales , Fenómenos Biomecánicos , Cabras , Masculino , Mitocondrias/efectos de los fármacos , Membranas Mitocondriales/efectos de los fármacos , Espermatozoides/efectos de los fármacosRESUMEN
The present study was attempted to investigate the dynamics of HSPA1A and redox status in the spermatozoa and fluid of different segments of buck epididymis. Testes were collected from sexually mature and healthy bucks aged between 2 and 3 years. The fluid and spermatozoa from different segments (caput, corpus and cauda) were harvested for further processing and analysis. The concentration of HSPA1A in spermatozoa lysate and epididymal fluid and its relative mRNA expression in spermatozoa from different segments of epididymis were studied. The HSPA1A concentration in epididymal fluid was significantly (P < 0.01) higher in the corpus as compared with caput and cauda, whereas, its concentration and relative mRNA expression decreased significantly (P < 0.01) in the spermatozoa from caput to cauda. The activities of SOD, GR, GST, and concentrations of manoldialdehyde and ROS decreased significantly (P < 0.01) in the spermatozoa from caput to cauda. The glutathione concentration and GPx activity decreased significantly (P < 0.01) in the spermatozoa of cauda as compared with the corpus. The SOD activity and ROS concentration were significantly (P < 0.01) higher in corpus, and GR and GST activity were significantly (P < 0.01) higher in caput fluid as compared with corpus and cauda. It may be concluded that HSPA1A concentration and its relative mRNA expression in spermatozoa decreased progressively, and redox status was altered during transit from caput to cauda.
Asunto(s)
Epidídimo/metabolismo , Cabras/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Espermatozoides/metabolismo , Animales , Líquidos Corporales/metabolismo , Supervivencia Celular , Cabras/genética , Proteínas HSP70 de Choque Térmico/genética , Masculino , Oxidación-Reducción , ARN Mensajero/metabolismo , Motilidad EspermáticaRESUMEN
Present study was undertaken on buck spermatozoa to investigate the effect of mercuric chloride on functional dynamics of buck spermatozoa. Four different concentrations (0.031, 0.125, 0.25 and 1.25 µg/mL) of mercuric chloride, which were 1/40th, 1/10th, 1/5th and equivalent to the LC50 value of HgCl2, were selected for studying their effect following in vitro exposure for 15 min and 3 h. Exposure of spermatozoa to 0.031 µg/mL mercuric chloride for 3 h resulted in significant (p < 0.05) decrease in sperm motility, sperm having intact membrane, intact acrosome and high mitochondrial trans-membrane potential. However, following exposure to higher concentrations (0.25, 1.25 µg/mL), similar results were observed even after 15 min of exposure. HgCl2 significantly (p < 0.05) increased the levels of malondialdehyde and reactive oxygen species and significantly (p < 0.05) decreased total antioxidant capacity and superoxide dismutase activity in spermatozoa within 15 min of exposure. Mercuric chloride-treated spermatozoa did not show capacitation, rather exhibited spontaneous acrosome reaction along with significant increase in intracellular Ca2+ and cAMP levels. Immuno-blotting of semen samples of control and 0.031 µg/mL mercury-treated groups showed low intensity bands of p55, p70, p80, p105 and p190 kDa tyrosine phosphorylation proteins while higher concentration-treated groups showed no such bands. Our findings evidently suggest that mercuric chloride even at 0.031 µg/mL adversely affected sperm functions, inhibited tyrosine phosphorylation proteins and capacitation due to oxidative stress. Spontaneous acrosome reaction (AR) in mercury-treated spermatozoa may possibly be due to increase in intracellular Ca2+ and cAMP levels, and capacitation failure may be due to inhibition of tyrosine phosphorylation of proteins.