Studies of in vitro models demonstrate that a forward motility protein (FMP) is required for the initiation of forward motility in the immature epididymal spermatozoa. FMP is a heat-stable glycoprotein derived from epididymal plasma. During the epididymal maturation of spermatozoa in vivo, there is a marked increase of intrasperm pH and level of cyclic adenosine monophosphate (CAMP). Several studies suggest that exogenous FMP in concert with elevated intrasperm pH and level of cAMP initiates flagellar motility during the epididymal transit of sperm. cAMP activates sperm cytosolic CAMPdependent protein kinases, which in turn phosphorylate multiple intrasperm phosphoproteins that may regulate flagellar motility. Exogenous calcium ion activates intact sperm motility, although it inhibits motility of demembranated cells on reactivation. Occurrence of CAMP-dependent type I and II protein kinases, a novel CAMP-independent protein kinase, and a phosphoprotein phosphatase has been demonstrated on the external surface of spermatozoa. The sperm surface has a coupled-enzyme system: ecto-CAMP-independent protein kinase and phosphoprotein phosphatase that regulate the phosphorylation and dephosphorylation of endogenous sperm ectophosphoproteins. The specific activities of these ectoenzymes increase markedly during forward progression, suggesting that they may have a role in regulating flagellar motility.
An improved Ficoll density gradient centrifugation method has been described for the isolation of goat caput-epididymal immature spermatozoa of a high purity and intactness. The method consists of layering freshly extracted sperm suspension on the top of a Ficoll gradient comprising 2.5 ml each of 2%, 4 % , 6%, and 8 % Ficoll-400 in a modified Ringer's solution and centrifugation at 300 g for 3 min in a swing bucket table centrifuge. Spermatozoa, free from fat globules and blood cells, sedimented at the bottom of the 8% Ficoll layer. The plasma membrane of the isolated cells showed a high degree of intactness (approximately 96%) as assessed by lactic dehydrogenase marker enzyme and ethidium bromide-fluorescence methods.
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