A Study of Rat Epididymal Sperm Adenosine 3′,5′-Monophosphate-Dependent Protein Kinases: Maturation Differences and Cellular Location

Abstract

The photoaffinity analog [32P]8-N3 cAMP (8-azido adenosine 3',5'-monophosphate was used to analyze the membrane sidedness of rat sperm cAMP binding proteins during epididymal maturation. Evidence is presented here which supports the hypothesis that 35-45% of the regulatory subunits of the Type I and Type II cAMP-dependent protein kinases are readily available to externally added cyclic nucleotide. It was observed by sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) and autoradiography that only two rat sperm proteins (Mr = 49K and 55K) were photolabeled which comigrated on gels with partially purified Type I and Type II regulatory subunits, respectively. Both of these photolabeled epididymal sperm proteins were saturated at physiological titers of [32P]8-N3cAMP and photoincorporation of [32P]8-N3 cAMP was specific since other SDS-resolvable sperm proteins did not photoincorporate the analog. Caput and cauda sperm protein photoincorporation could be effectively blocked by low levels of cAMP, but not by cGMP, ATP or GTP. Sperm epididymal maturation coincided with changes in the cAMP-dependent protein kinase subunits since cauda sperm contained more available Type II than did caput sperm. A subcellular analysis of cAMP-dependent protein kinase regulatory subunit in head and tail fractions was done for caput and cauda sperm and demonstrated that the tail fractions showed more photo-labeling of both Type I and II regulatory subunits than did the head fractions.

Biochemical Parameters of Initiation and Regulation of Sperm Motility

“…The primary bases of this conclusion with rat epididymal sperm are that approximately 30% of the cells are leaky, as shown by assays of cytosolic enzyme markers, and that all the CAMP-binding proteins are available to externally added 8-[32P]-N, cAMP and a-chymotrypsin. In contrast to this report, Atherton et al [4] observed that approximately 60% of CAMP-binding proteins of intact spermatozoa require sonication to be readily available to 8-[32P]--N, cAMP or proteolytic enzymes. They also confirmed the intactness of spermatozoa by assaying hexokinase as a marker enzyme [4].…”

“…In contrast to this report, Atherton et al [4] observed that approximately 60% of CAMP-binding proteins of intact spermatozoa require sonication to be readily available to 8-[32P]--N, cAMP or proteolytic enzymes. They also confirmed the intactness of spermatozoa by assaying hexokinase as a marker enzyme [4].…”

“…CAMP-dependent phosphorylation of proteins of different molecular weights takes place in ram sperm of different maturational status [132]. The mature (cauda epididymidis) rat sperm contains a higher level of a CAMP-dependent protein kinase compared to the immature (caput epididymidis) sperm [4]. The precise role of CAMP-dependent protein kinase and protein phosphorylation with sperm maturation and motility cannot be ascertained, however.…”

Membrane-associated protein kinase activities in seminal plasma from vasectomized men

“…Protein kinase activities in semen are associated with both spermatozoa (Hoskins et al, 1972;Garbers et al, 1973;Majumder, 1978a;Tash & Means, 1982;Horowitz et al, 1984;Atherton et al, 1985) and seminal plasma (Majumder, 1978b;Fabbro et al, 1982;. Approxi¬ mately 70-80% of the protein kinase activities in seminal plasma are correlated with the number of spermatozoa originally present in the ejaculate (Fabbro et al, 1982;Wilson & Kaye, 1983).…”

Azido cAMP, [32P]8N3cAMP: A Probe to Study Epididymal Sperm Maturation

“…Specifically, the azido cAMP analogue ['2P]8N3cAMP is an excellent probe for the study of epididyma1 sperm maturation [2]. Since epididymal sperm maturation confers fertilizing ability on sperm, the process is important to both contraceptive and infertility research.…”