Lead and calcium crosstalk tempted acrosome damage and hyperpolarization of spermatozoa: signaling and ultra-structural evidences.

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.

Quercetin in semen extender curtails reactive oxygen and nitrogen species and improves functional attributes of cryopreserved buck semen.

Cryopreservation of goat spermatozoa is challenging due to several factors, including one of the most essential, i.e., oxidative stress. It is particularly essential in goat semen due to its scanty ejaculate volume and high sperm concentration. This leaves a narrow sperm-to-seminal plasma ratio owing to marginal antioxidant support; moreover, semen extension further dilutes the antioxidant level, leading to an imbalance of oxidant-antioxidant equilibrium. The present study aimed to evaluate the effect of quercetin on curtailing oxidative stress and its reflection on the post-thaw survivability and membrane integrity of goat spermatozoa. For this study, six bucks were selected. Six ejaculates from each buck totaling 36 ejaculates were collected, which were then split into five parts; furthermore, each part was added with a semen extender having a particular concentration of additive. Group C without quercetin and T1 containing Vitamin E at 3 mmol/mL were considered the control and positive control respectively, whereas T2, T3, and T4 contain 10, 20, and 30 µmol/mL of Quercetin respectively. The final sperm concentration of each group was kept at 200 × 106 spermatozoa/mL. All groups were subjected to equilibration at 4 °C for 4 h, then filled in French mini (0.25 mL) straws, followed by sealing and cryopreservation. Samples after 72 h of cryopreservation were subjected to evaluation of plasma membrane integrity and viability through staining, acrosomal integrity, and mitochondrial membrane activity through flow cytometry. Evaluation of sperm kinematics as well as the oxidant-antioxidant status of sperm (ROS and nitric oxide) and seminal plasma (SOD, CAT, GPx, FRAP, and lipid peroxidation through MDA estimation) were also carried out. Quercetin, when supplemented at 20 µmol/mL in buck semen extender, significantly (p < 0.01) improved cryopreserved sperm functions in terms of plasma membrane integrity, viability, acrosomal integrity, mitochondrial membrane activity, and sperm kinematics of buck semen. Similarly, Quercetin supplementation at 20 µmol/mL significantly reduced reactive oxygen and nitrogen species (RONS) in sperm and improved the antioxidant status of seminal plasma, which was indicated by reduced oxidative damage and improved the antioxidant status of buck semen. In conclusion, Quercetin at 20 µmol/mL reduced oxidative stress, improved semen antioxidant status, and improved sperm membranes integrity and kinematics.

Freezing-thawing induces deprotamination, cryocapacitation-associated changes; DNA fragmentation; and reduced progesterone sensitivity in buck spermatozoa.

In the present study, there was evaluation of cryocapacitation-associated changes, apoptotic-like changes, deprotamination, total antioxidant capacity (TAC), and in vitro sperm functional attributes in Barbari bucks after freezing-thawing. The correlation between deprotamination and sperm functional characteristics was established. Using immunoblotting procedures, there was detection of the presence of a single 28-kDa protein band corresponding to protamine-1. The localization in the head region of the spermatozoa was further validated by an immunofluorescence test. Capacitated (B-) and acrosome-reacted (AR-) pattern spermatozoa, spermatozoa with the externalization of phosphatidylserine and a relatively lesser mitochondrial transmembrane potential, and deprotamination and DNA fragmentation was greater (P < 0.05) after freezing-thawing and indicated there were cryocapacitation- and apoptotic-like changes, respectively. Furthermore, the detection of phosphorylation of tyrosine-containing proteins with use of immunoblotting and immunofluorescence procedures confirmed there were cryocapacitation-like changes in the buck spermatozoa after freezing-thawing. Total antioxidant capacity (TAC), in vitro thermal resistance response, Vanguard distance, progesterone sensitivity, and in vitro capacitation response were less (P < 0.05) in the spermatozoa after freezing-thawing compared with spermatozoa after initial dilution and equilibration. Deprotamination (chromomycin A3-positive cells, CMA3+) and DNA fragmentation (TUNEL+ve) were positively correlated with B- and AR-pattern spermatozoa, while other values for other variables were negatively correlated. In conclusion, the results of this study indicated there was protamine-1 in buck spermatozoa and after freezing-thawing there was a loss of protamine-1 combined with cryocapacitation-associated changes and apoptotic-like changes in buck spermatozoa. Spermatozoa deprotamination might be attributed to increased DNA fragmentation, resulting in compromised fertilizing capacity of buck spermatozoa.