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Cell-free enzyme preparations consisting of lipoglycoprotein were obtained from acrosomes of ram, bull and rabbit spermatozoa. The ram and bull preparations showed proteolytic and hyaluronidase activities. Preparations from the three species brought about dispersal of the cumulus oophorus and corona radiata of newly ovulated rabbit eggs. In some cases the zona pellucida was also removed. Iodoacetate and a polyanionic hyaluronidase inhibitor each reduced the denuding activity of the ram preparation and to a lesser extent of the bull preparation. Heating to 100\s=deg\C also reduced the activity of the ram and bull preparations. It is concluded that proteolytic enzymes in spermatozoa contribute to the denudation of rabbit eggs and probably facilitate penetration of the zona pellucida.

A method involving the treatment of ram spermatozoa with MgCl2 followed by Hyamine 2389 and Triton X-100 is described, which gave selective removal of the acrosomal membranes and acrosomal enzymes. The effect on the individual membranes of ram spermatozoa was evaluated by electron microscopy. Treatment with MgCl2 removed or altered the integrity of the plasma and outer acrosomal membranes, allowing the release of material from the acrosome. The inner acrosomal membrane and the electron-dense material of the equatorial segment were frequently unaffected by this treatment. Subsequent treatment of these spermatozoa with Hyamine and Triton removed the inner acrosomal membrane and the electron-dense material from the equatorial segment. The MgCl2 extract contained acrosomal proteinases and hyaluronidase. The detergent extract contained sperm neuraminidase. The specific activities and yields of the enzymes obtained in the MgCl2 step were much higher than those of the enzymes obtained by the detergent treatment. Slight alterations in the conditions of the treatment had different effects on the acrosomal membranes in that one or more of the membranes were removed or altered in varying proportions resulting in the release of one or more enzymes. Only the plasma membrane and the acrosome appeared to be affected by these treatments as selected mitochondrial enzymes were not detectable in the extracts.

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With the increasing evidences of cadmium-induced cognitive deficits associated with brain cholinergic dysfunctions, the present study aimed to decipher molecular mechanisms involved in the neuroprotective efficacy of quercetin in rats. A decrease in the binding of cholinergic-muscarinic receptors and mRNA expression of cholinergic receptor genes (M1, M2, and M4) was observed in the frontal cortex and hippocampus on exposure of rats to cadmium (5.0 mg/kg body weight, p.o.) for 28 days compared to controls. Cadmium exposure resulted to decrease mRNA and protein expressions of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) and enhance reactive oxygen species (ROS) generation associated with mitochondrial dysfunctions, ultrastructural changes, and learning deficits. Enhanced apoptosis, as evidenced by alterations in key proteins involved in the pro- and anti-apoptotic pathway and mitogen-activated protein (MAP) kinase signaling, was evident on cadmium exposure. Simultaneous treatment with quercetin (25 mg/kg body weight, p.o.) resulted to protect cadmium-induced alterations in cholinergic-muscarinic receptors, mRNA expression of genes (M1, M2, and M4), and expression of ChAT and AChE. The protective effect on brain cholinergic targets was attributed to the antioxidant potential of quercetin, which reduced ROS generation and protected mitochondrial integrity by modulating proteins involved in apoptosis and MAP kinase signaling. The results exhibit that quercetin may modulate molecular targets involved in brain cholinergic signaling and attenuate cadmium neurotoxicity.

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