cDNA clones coding for two closely related androgen-dependent sperm-coating glycoproteins secreted by the rat epididymis were selected by screening an epididymal cDNA library constructed in Agt 11 with affinity-purified antibody directed against the glycoproteins. The largest clone of 956 nucleotides provided coding information for a protein of 246 amino acids of which the first 19 residues comprise a putative signal peptide sequence which when cleaved would produce a mature protein of 227 residues and a molecular mass of 26 kDa. Confirmation of the identity of the clone was provided by a match between the amino acid sequence predicted from the cDNA sequence and the actual amino acid sequence determined for a tryptic peptide fragment of one of the pure glycoproteins. It is probable that the primary amino acid sequence of the two glycoproteins is identical. Northern blot and slot-blot analysis revealed that the mRNA for the glycoproteins is approximately 1250 nucleotides long and that the concentration of the mRNA in the epididymis is androgen-dependent. The glycoproteins and their mRNAs were unique to the epididymis as determined by Western and Northern blots, respectively, since signals were absent from skin, brain, liver, kidney, heart, skeletal muscle and testis. Cross-reacting proteins of slightly smaller apparent molecular mass were detected in extracts of mouse and guinea-pig epididymis, but not rabbit or bull epididymis. Comparison with existing protein data bases revealed that the epididymal glycoproteins display significant sequence homology with yeast carboxypeptidase Y.The mammalian epididymis is a single long convoluted tubule which conveys spermatozoa from the testis to the ductus deferens. During passage through this tubule spermatozoa undergo functional maturation which is manifest in the progressive acquisition by the cells of the ability to swim [l, 21, to bind to the zona pellucida of the oocyte [3, 41, and to effect fertilization [5]. A number of studies indicate that functional maturation is not an intrinsic property of the sperm, but is brought about by an interaction of sperm with a variety of androgen-dependent proteins which are secreted by the epididymal epithelium and then become associated with the sperm surface [6-131. We have recently reported the cDNA cloning and primary amino acid sequence of two androgen-dependent epididymal secretory proteins of 18.5 kDa which are thought to be involved in this way in sperm development [ 141. However, particular attention has also been focused on some acidic glycoproteins which have been given several names such as proteins D and E [15, 161, acidic epididymal glycoprotein [7], protein IV [17], sialoprotein [18], and 32 K protein [19]. The glycoproteins have been variously reported to have isoelectric points between 4.0 and 5.1 and molecular masses in the range of 27-37 kDa

Spermatozoa from the testis and cauda epididymidis of the rat were surface labelled with radioactive iodide. Detergent extracts of radioiodinated spermatozoa immunoprecipitated with antisera against specific epididymal proteins, followed by polyacrylamide gel electrophoresis, revealed two proteins (D and E of Mr 27 000 and 28 000, respectively) which became associated with spermatozoa during epididymal transit. These proteins were observed by immunofluorescence microscopy to be located over a restricted area of the head surface. Proteins with similar molecular weight were labelled on spermatozoa from the cauda epididymidis, but not from the testis, by reaction with sodium boro[3H]hydride in the presence of galactose oxidase. However, failure to immunoprecipitate with antibodies to Proteins D and E and non-coincident migration on two-dimensional gel electrophoresis established the non-identity of these proteins. Compared with Proteins D and E, two other major epididymal secretory proteins (Proteins B and C of Mr 16 000) associated with spermatozoa to a relatively minor extent during epididymal transit.

Summary. Spermatozoa from the testis and cauda epididymidis of the rat were surface labelled with radioactive iodide, extracted with detergent, and the radioactive proteins separated by two-dimensional

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